Silent Phase of Johne’s Disease in Experimentally Infected Goats – A Study on New and Established Diagnostic Approaches Using Specific and Non-Specific Parameters

The current gold standard diagnostic test for Johne’s disease (JD) is detecting Mycobacterium avium subsp. paratuberculosis (MAP) from fecal samples via culture and/or PCR. Other commercially available JD diagnostic tests focus on the detection of specific antibodies within the serum or milk of infected ruminants. These tests have a high specificity but low their sensitivity and usually fail to diagnose the disease until later stages of the disease. The ideal diagnostic test should detect infected animals already during the silent phase. Here, we evaluate the use of new and established approaches to define the silent phase of JD in experimentally infected goats. None of the established diagnostic tests or new approaches for the detection of humoral and cellular immune responses were positive during the first year of infection. Only the characterization of various subsets of peripheral blood leukocytes and the weight development gave some indication for the presence of a chronic, but silent, infection. Weight differences were present throughout the first year. In addition, some of the subsets of leukocytes (WC1+ T cells, MHC class II+ leukocytes, CD1+ leukocytes, CD14+ granulocytes, and CD14+/MHC class II+ granulocytes) demonstrated significant differences, but only at certain time points

Functional drug screening reveals anticonvulsants as enhancers of mTOR-independent autophagic killing of Mycobacterium tuberculosis through inositol depletion.

Mycobacterium tuberculosis (MTB) remains a major challenge to global health made worse by the spread of multidrug resistance. We therefore examined whether stimulating intracellular killing of mycobacteria through pharmacological enhancement of macroautophagy might provide a novel therapeutic strategy. Despite the resistance of MTB to killing by basal autophagy, cell-based screening of FDA-approved drugs revealed two anticonvulsants, carbamazepine and valproic acid, that were able to stimulate autophagic killing of intracellular M. tuberculosis within primary human macrophages at concentrations achievable in humans. Using a zebrafish model, we show that carbamazepine can stimulate autophagy in vivo and enhance clearance of M. marinum, while in mice infected with a highly virulent multidrug-resistant MTB strain, carbamazepine treatment reduced bacterial burden, improved lung pathology and stimulated adaptive immunity. We show that carbamazepine induces antimicrobial autophagy through a novel, evolutionarily conserved, mTOR-independent pathway controlled by cellular depletion of myo-inositol. While strain-specific differences in susceptibility to in vivo carbamazepine treatment may exist, autophagy enhancement by repurposed drugs provides an easily implementable potential therapy for the treatment of multidrug-resistant mycobacterial infection

Latency Antigen α-Crystallin Based Vaccination Imparts a Robust Protection against TB by Modulating the Dynamics of Pulmonary Cytokines

BACKGROUND: Efficient control of tuberculosis (TB) requires development of strategies that can enhance efficacy of the existing vaccine Mycobacterium bovis Bacille Calmette Guerin (BCG). To date only a few studies have explored the potential of latency-associated antigens to augment the immunogenicity of BCG. METHODS/PRINCIPAL FINDINGS: We evaluated the protective efficacy of a heterologous prime boost approach based on recombinant BCG and DNA vaccines targeting α-crystallin, a prominent latency antigen. We show that "rBCG prime-DNA boost" strategy (R/D) confers a markedly superior protection along with reduced pathology in comparison to BCG vaccination in guinea pigs (565 fold and 45 fold reduced CFU in lungs and spleen, respectively, in comparison to BCG vaccination). In addition, R/D regimen also confers enhanced protection in mice. Our results in guinea pig model show a distinct association of enhanced protection with an increased level of interleukin (IL)12 and a simultaneous increase in immuno-regulatory cytokines such as transforming growth factor (TGF)β and IL10 in lungs. The T cell effector functions, which could not be measured in guinea pigs due to technical limitations, were characterized in mice by multi-parameter flow cytometry. We show that R/D regimen elicits a heightened multi-functional CD4 Th1 cell response leading to enhanced protection. CONCLUSIONS/SIGNIFICANCE: These results clearly indicate the superiority of α-crystallin based R/D regimen over BCG. Our observations from guinea pig studies indicate a crucial role of IL12, IL10 and TGFβ in vaccine-induced protection. Further, characterization of T cell responses in mice demonstrates that protection against TB is predictable by the frequency of CD4 T cells simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and IL2. We anticipate that this study will not only contribute toward the development of a superior alternative to BCG, but will also stimulate designing of TB vaccines based on latency antigens

Alternative BCG delivery strategies improve protection against Mycobacterium tuberculosis in non-human primates: Protection associated with mycobacterial antigen-specific CD4 effector memory T-cell populations

Intradermal (ID) BCG injection provides incomplete protection against TB in humans and experimental models. Alternative BCG vaccination strategies may improve protection in model species, including rhesus macaques. This study compares the immunogenicity and efficacy of BCG administered by ID and intravenous (IV) injection, or as an intratracheal mucosal boost (ID + IT), against aerosol challenge with Mycobacterium tuberculosis Erdman strain. Disease pathology was significantly reduced, and survival improved, by each BCG vaccination strategy, relative to unvaccinated animals. However, IV induced protection surpassed that achieved by all other routes, providing an opportunity to explore protective immunological mechanisms using antigen-specific IFN-γ ELISpot and polychromatic flow cytometry assays. IFN-γ spot forming units and multifunctional CD4 T-cell frequencies increased significantly following each vaccination regimen and were greatest following IV immunisation. Vaccine-induced multifunctional CD4 T-cells producing IFN-γ and TNF-α were associated with reduced disease pathology following subsequent M.tb challenge; however, high frequencies of this population following M.tb infection correlated with increased pathology. Cytokine producing T-cells primarily occupied the CD4 transitional effector memory phenotype, implicating this population as central to the mycobacterial response, potentially contributing to the stringent control observed in IV vaccinated animals. This study demonstrates the protective efficacy of IV BCG vaccination in rhesus macaques, offering a valuable tool for the interrogation of immunological mechanisms and potential correlates of protection

Increased Virulence of an Epidemic Strain of Mycobacterium massiliense in Mice

Chronic pulmonary disease and skin/soft tissue infections due to non-tuberculous mycobacteria (NTM) of the Mycobacterium chelonae-abscessus-massiliense group is an emerging health problem worldwide. Moreover, the cure rate for the infections this group causes is low despite aggressive treatment. Post-surgical outbreaks that reached epidemic proportions in Brazil recently were caused by M. massiliense isolates resistant to high-level disinfection with glutaraldehyde (GTA). Understanding the differences in the virulence and host immune responses induced by NTM differing in their sensitivity to disinfectants, and therefore their relative threat of causing outbreaks in hospitals, is an important issue.We compared the replication and survival inside macrophages of a GTA-susceptible reference Mycobacterium massiliense clinical isolate CIP 108297 and an epidemic strain from Brazil, CRM-0019, and characterized the immune responses of IFNγ knockout mice exposed to a high dose aerosol with these two isolates. CRM-0019 replicated more efficiently than CIP 108297 inside mouse bone marrow macrophages. Moreover, the animals infected with CRM-0019 showed a progressive lung infection characterized by a delayed influx of CD4+ and CD8+ T cells, culminating in extensive lung consolidation and demonstrated increased numbers of pulmonary CD4+ Foxp3+ regulatory T cells compared to those infected with the reference strain. Immunosuppressive activity of regulatory T cells may contribute to the progression and worsening of NTM disease by preventing the induction of specific protective immune responses.These results provide the first direct evidence of the increased virulence in macrophages and mice and pathogenicity in vivo of the Brazilian epidemic isolate and the first observation that NTM infections can be associated with variable levels of regulatory T cells which may impact on their virulence and ability to persist in the host

Enhanced upper genital tract pathologies by blocking Tim-3 and PD-L1 signaling pathways in mice intravaginally infected with Chlamydia muridarum

<p>Abstract</p> <p>Background</p> <p>Although Tim-3 & PD-L1 signaling pathways play important roles in negatively regulating immune responses, their roles in chlamydial infection have not been evaluated.</p> <p>Methods</p> <p>Neutralization antibodies targeting Tim-3 and PD-L1 were used to treat mice. Following an intravaginal infection with <it>C. muridarum </it>organisms, mice with or without the dual antibody treatment were compared for live chlamydial organism shedding from the lower genital tract and inflammatory pathology in the upper genital tract.</p> <p>Results</p> <p>Mice treated with anti-Tim-3 and anti-PD-L1 antibodies displayed a time course of live organism shedding similar to that of mice treated with equivalent amounts of isotype-matched IgG molecules. The combined antibody blocking failed to alter either the lower genital tract cytokine or systemic humoral and cellular adaptive responses to <it>C. muridarum </it>infection. However, the antibody blocking significantly enhanced <it>C. muridarum</it>-induced pathologies in the upper genital tract, including more significant hydrosalpinx and inflammatory infiltration in uterine horn and oviduct tissues.</p> <p>Conclusions</p> <p>The Tim-3 and PD-L1-mediated signaling can significantly reduce pathologies in the upper genital tract without suppressing immunity against chlamydial infection, suggesting that Tim-3 and PD-L1-mediated negative regulation may be manipulated to attenuate tubal pathologies in women persistently infected with <it>C. trachomatis </it>organisms.</p

A higher activation threshold of memory CD8+ T cells has a fitness cost that is modified by TCR affinity during Tuberculosis

All relevant data are within the paper and its Supporting Information files except for the primary TCR sequences. The data files for the primary TCR sequences are publicly deposited in the University of Massachusetts Medical School’s institutional repository, eScholarship@UMMS. The permanent link to the data is http://dx.doi.org/10.13028/M2CC70T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.This work was supported by NIH R01 AI106725 as well as fellowship funding to SC from NIH AI T32 007061 and the UMass GSBS Millennium Program. The Small Animal Biocontainment Suite was supported in part by Center for AIDS Research Grant P30 AI 060354. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio

Trying to See the Forest through the Trees: Deciphering the Nature of Memory Immunity to Mycobacterium tuberculosis

The purpose of vaccination against tuberculosis and other diseases is to establish a heightened state of acquired specific resistance in which the memory immune response is capable of mediating an accelerated and magnified expression of protection to the pathogen when this is encountered at a later time. In the earliest studies in mice infected with Mycobacterium tuberculosis, memory immunity and the cells that express this were definable both in terms of kinetics of emergence, and soon thereafter by the levels of expression of markers including CD44, CD62L, and the chemokine receptor CCR7, allowing the identification of effector memory and central memory T cell subsets. Despite these initial advances in knowledge, more recent information has not revealed more clarity, but instead, has created a morass of complications—complications that, if not resolved, could harm correct vaccine design. Here, we discuss two central issues. The first is that we have always assumed that memory is induced in the same way, and consists of the same T cells, regardless of whether that immunity is generated by BCG vaccination, or by exposure to M. tuberculosis followed by effective chemotherapy. This assumption is almost certainly incorrect. Second, a myriad of additional memory subsets have now been described, such as resident, stem cell-like, tissue specific, among others, but as yet we know nothing about the relative importance of each, or whether if a new vaccine needs to induce all of these, or just some, to be fully effective

Gr1(int)CD11b+ myeloid-derived suppressor cells in Mycobacterium tuberculosis infection.

Tuberculosis is one of the world's leading killers, stealing 1.4 million lives and causing 8.7 million new and relapsed infections in 2011. The only vaccine against tuberculosis is BCG which demonstrates variable efficacy in adults worldwide. Human infection with Mycobacterium tuberculosis results in the influx of inflammatory cells to the lung in an attempt to wall off bacilli by forming a granuloma. Gr1(int)CD11b(+) cells are called myeloid-derived suppressor cells (MDSC) and play a major role in regulation of inflammation in many pathological conditions. Although MDSC have been described primarily in cancer their function in tuberculosis remains unknown. During M. tuberculosis infection it is crucial to understand the function of cells involved in the regulation of inflammation during granuloma formation. Understanding their relative impact on the bacilli and other cellular phenotypes is necessary for future vaccine and drug design.We compared the bacterial burden, lung pathology and Gr1(int)CD11b(+) myeloid-derived suppressor cell immune responses in M. tuberculosis infected NOS2-/-, RAG-/-, C3HeB/FeJ and C57/BL6 mice. Gr-1(+) cells could be found on the edges of necrotic lung lesions in NOS2-/-, RAG-/-, and C3HeB/FeJ, but were absent in wild-type mice. Both populations of Gr1(+)CD11b(+) cells expressed high levels of arginase-1, and IL-17, additional markers of myeloid derived suppressor cells. We then sorted the Gr1(hi) and Gr1(int) populations from M. tuberculosis infected NOS-/- mice and placed the sorted both Gr1(int) populations at different ratios with naïve or M. tuberculosis infected splenocytes and evaluated their ability to induce activation and proliferation of CD4+T cells. Our results showed that both Gr1(hi) and Gr1(int) cells were able to induce activation and proliferation of CD4+ T cells. However this response was reduced as the ratio of CD4(+) T to Gr1(+) cells increased. Our results illustrate a yet unrecognized interplay between Gr1(+) cells and CD4(+) T cells in tuberculosis