Antibiotic-Loaded Polymersomes for Clearance of Intracellular Burkholderia thailandensis.

Melioidosis caused by the facultative intracellular pathogen Burkholderia pseudomallei is difficult to treat due to poor intracellular bioavailability of antibiotics and antibiotic resistance. In the absence of novel compounds, polymersome (PM) encapsulation may increase the efficacy of existing antibiotics and reduce antibiotic resistance by promoting targeted, infection-specific intracellular uptake. In this study, we developed PMs composed of widely available poly(ethylene oxide)-polycaprolactone block copolymers and demonstrated their delivery to intracellular B. thailandensis infection using multispectral imaging flow cytometry (IFC) and coherent anti-Stokes Raman scattering microscopy. Antibiotics were tightly sequestered in PMs and did not inhibit the growth of free-living B. thailandensis. However, on uptake of antibiotic-loaded PMs by infected macrophages, IFC demonstrated PM colocalization with intracellular B. thailandensis and a significant inhibition of their growth. We conclude that PMs are a viable approach for the targeted antibiotic treatment of persistent intracellular Burkholderia infection.

Protection induced by a Francisella tularensis subunit vaccine delivered by glucan particles.

Francisella tularensis is an intracellular pathogen causing the disease tularemia, and an organism of concern to biodefence. There is no licensed vaccine available. Subunit approaches have failed to induce protection, which requires both humoral and cellular immune memory responses, and have been hampered by a lack of understanding as to which antigens are immunoprotective. We undertook a preliminary in silico analysis to identify candidate protein antigens. These antigens were then recombinantly expressed and encapsulated into glucan particles (GPs), purified Saccharomyces cerevisiae cell walls composed primarily of ß-1,3-glucans. Immunological profiling in the mouse was used to down-selection to seven lead antigens: FTT1043 (Mip), IglC, FTT0814, FTT0438, FTT0071 (GltA), FTT0289, FTT0890 (PilA) prior to transitioning their evaluation to a Fischer 344 rat model for efficacy evaluation. F344 rats were vaccinated with the GP protein antigens co-delivered with GP-loaded with Francisella LPS. Measurement of cell mediated immune responses and computational epitope analysis allowed down-selection to three promising candidates: FTT0438, FTT1043 and FTT0814. Of these, a GP vaccine delivering Francisella LPS and the FTT0814 protein was able to induce protection in rats against an aerosol challenge of F. tularensis SchuS4, and reduced organ colonisation and clinical signs below that which immunisation with a GP-LPS alone vaccine provided. This is the first report of a protein supplementing protection induced by LPS in a Francisella vaccine. This paves the way for developing an effective, safe subunit vaccine for the prevention of inhalational tularemia, and validates the GP platform for vaccine delivery where complex immune responses are required for prevention of infections by intracellular pathogens.

Increased TNF-α/IFN-γ/IL-2 and Decreased TNF-α/IFN-γ Production by Central Memory T Cells Are Associated with Protective Responses against Bovine Tuberculosis Following BCG Vaccination.

Central memory T cell (Tcm) and polyfunctional CD4 T cell responses contribute to vaccine-elicited protection with both human and bovine tuberculosis (TB); however, their combined role in protective immunity to TB is unclear. To address this question, we evaluated polyfunctional cytokine responses by CD4 T cell effector/memory populations from bacille Calmette-Guerin (BCG) vaccinated and non-vaccinated calves by flow cytometry prior to and after aerosol challenge with virulent Mycobacterium bovis. Polyfunctional cytokine expression patterns in the response by Tcm, effector memory, and effector T cell subsets were similar between BCG-vaccinated and M. bovis-infected calves, only differing in magnitude (i.e., infected > vaccinated). BCG vaccination, however, did alter the kinetics of the ensuing response to virulent M. bovis infection. Early after challenge (3 weeks post-infection), non-vaccinates had greater antigen-specific interferon-γ (IFN-γ)/tumor necrosis factor-α (TNF-α) and lesser IFN-γ/TNF-α/IL-2 responses by Tcm cells than did vaccinated animals. Importantly, these differences were also associated with mycobacterial burden upon necropsy. Polyfunctional responses to ESAT-6:CFP10 (antigens not synthesized by BCG strains) were detected in memory subsets, as well as in effector cells, as early as 3 weeks after challenge. These findings suggest that cell fate divergence may occur early after antigen priming in the response to bovine TB and that memory and effector T cells may expand concurrently during the initial phase of the immune response. In summary, robust IFN-γ/TNF-α response by Tcm cells is associated with greater mycobacterial burden, while IFN-γ/TNF-α/IL-2 response by Tcm cells are indicative of a protective response to bovine TB.

Protection Induced by Simultaneous Subcutaneous and Endobronchial Vaccination with BCG/BCG and BCG/Adenovirus Expressing Antigen 85A against Mycobacterium bovis in Cattle.

The incidence of bovine tuberculosis (bTB) in the GB has been increasing since the 1980s. Immunisation, alongside current control measures, has been proposed as a sustainable measure to control bTB. Immunisation with Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been shown to protect against bTB. Furthermore, much experimental data indicates that pulmonary local immunity is important for protection against respiratory infections including Mycobacterium tuberculosis and that pulmonary immunisation is highly effective. Here, we evaluated protection against M. bovis, the main causative agent of bTB, conferred by BCG delivered subcutaneously, endobronchially or by the new strategy of simultaneous immunisation by both routes. We also tested simultaneous subcutaneous immunisation with BCG and endobronchial delivery of a recombinant type 5 adenovirus expressing mycobacterial antigen 85A. There was significantly reduced visible pathology in animals receiving the simultaneous BCG/BCG or BCG/Ad85 treatment compared to naïve controls. Furthermore, there were significantly fewer advanced microscopic granulomata in animals receiving BCG/Ad85A compared to naive controls. Thus, combining local and systemic immunisation limits the development of pathology, which in turn could decrease bTB transmission.

Development of an antibody to bovine IL-2 reveals multifunctional CD4 T(EM) cells in cattle naturally infected with bovine tuberculosis.

Gaining a better understanding of the T cell mechanisms underlying natural immunity to bovine tuberculosis would help to identify immune correlates of disease progression and facilitate the rational design of improved vaccine and diagnostic strategies. CD4 T cells play an established central role in immunity to TB, and recent interest has focussed on the potential role of multifunctional CD4 T cells expressing IFN-γ, IL-2 and TNF-α. Until now, it has not been possible to assess the contribution of these multifunctional CD4 T cells in cattle due to the lack of reagents to detect bovine IL-2 (bIL-2). Using recombinant phage display technology, we have identified an antibody that recognises biologically active bIL-2. Using this antibody, we have developed a polychromatic flow cytometric staining panel that has allowed the investigation of multifunctional CD4 T-cells responses in cattle naturally infected with M. bovis. Assessment of the frequency of antigen specific CD4 T cell subsets reveals a dominant IFN-γ(+)IL-2(+)TNF-α(+) and IFN-γ(+) TNF-α(+) response in naturally infected cattle. These multifunctional CD4 T cells express a CD44(hi)CD45RO(+)CD62L(lo) T-effector memory (T(EM)) phenotype and display higher cytokine median fluorescence intensities than single cytokine producers, consistent with an enhanced 'quality of response' as reported for multifunctional cells in human and murine systems. Through our development of these novel immunological bovine tools, we provide the first description of multifunctional T(EM) cells in cattle. Application of these tools will improve our understanding of protective immunity in bovine TB and allow more direct comparisons of the complex T cell mediated immune responses between murine models, human clinical studies and bovine TB models in the future.

Non-acylated Mycobacterium bovis glycoprotein MPB83 binds to TLR1/2 and stimulates production of matrix metalloproteinase 9.

A variety of Mycobacterium tuberculosis cell wall components induce expression of matrix metalloproteinase 9 (MMP-9) by monocytic cells and levels of MMP-9 in vivo positively correlate with severity of disease. Toll-like receptor (TLR)2 mediates cellular responses to acylated molecules but can also mediate responsiveness to diverse molecular structures, including non-acylated native viral and bacterial proteins. MPT/B-83 is a cell-associated lipoglycoprotein common to M. tuberculosis and M. bovis and an important antigen during infection of cattle. Since MPB83 is acylated and glycosylated, we investigated whether MPB83 would induce MMP-9 expression via interaction with TLR2, and assessed the contribution of the lipid, glycan and polypeptide components to its activity. Acylated peptide derived from MPB83 stimulated MMP-9 expression by human macrophage cells via interaction with both TLR2 and TLR1, but not TLR4. Lesser induction was found with secreted (non-acylated, but glycosylated) MPB83 protein purified from culture of M. bovis. Stimulation of cells with MPB83 induced TNF-α production which acted to upregulate MMP-9 expression. Surprisingly, recombinant MPB83 protein devoid of any post-translational modification also induced MMP-9 expression. Direct interaction of RecMPB83 with TLR2 was demonstrated by surface plasmon-resonance. MPB83 may act as a virulence factor through TLR2 mediated induction of MMP-9.

Revisiting host preference in the Mycobacterium tuberculosis complex: experimental infection shows M. tuberculosis H37Rv to be avirulent in cattle.

Experiments in the late 19th century sought to define the host specificity of the causative agents of tuberculosis in mammals. Mycobacterium tuberculosis, the human tubercle bacillus, was independently shown by Smith, Koch, and von Behring to be avirulent in cattle. This finding was erroneously used by Koch to argue the converse, namely that Mycobacterium bovis, the agent of bovine tuberculosis, was avirulent for man, a view that was subsequently discredited. However, reports in the literature of M. tuberculosis isolation from cattle with tuberculoid lesions suggests that the virulence of M. tuberculosis for cattle needs to be readdressed. We used an experimental bovine infection model to test the virulence of well-characterized strains of M. tuberculosis and M. bovis in cattle, choosing the genome-sequenced strains M. tuberculosis H37Rv and M. bovis 2122/97. Cattle were infected with approximately 10(6) CFU of M. tuberculosis H37Rv or M. bovis 2122/97, and sacrificed 17 weeks post-infection. IFN-gamma and tuberculin skin tests indicated that both M. bovis 2122 and M. tuberculosis H37Rv were equally infective and triggered strong cell-mediated immune responses, albeit with some indication of differential antigen-specific responses. Postmortem examination revealed that while M. bovis 2122/97-infected animals all showed clear pathology indicative of bovine tuberculosis, the M. tuberculosis-infected animals showed no pathology. Culturing of infected tissues revealed that M. tuberculosis was able to persist in the majority of animals, albeit at relatively low bacillary loads. In revisiting the early work on host preference across the M. tuberculosis complex, we have shown M. tuberculosis H37Rv is avirulent for cattle, and propose that the immune status of the animal, or genotype of the infecting bacillus, may have significant bearing on the virulence of a strain for cattle. This work will serve as a baseline for future studies into the genetic basis of host preference, and in particular the molecular basis of virulence in M. bovis.

Simple and effective technical procedures for the packing and application of nanospray PicoFrit HPLC columns for proteomic analyses.

A simple procedure was developed for packing PicoFrit HPLC columns with chromatographic stationary phase using a reservoir fabricated from standard laboratory HPLC fittings. Packed columns were mounted onto a stainless steel ultra-low volume precolumn filter assembly containing a 0.5-microm pore size steel frit. This format provided a conduit for the application of the nanospray voltage and protected the column from obstruction by sample material. The system was characterised and operational performance assessed by analysis of a range of peptide standards (n = 9).

Maturation of bovine dendritic cells by lipopeptides.

The response of DC, and the subsequent stimulation of T cells, is an essential part of the initiation of immune responses following microbial challenge. The response of human DC to bacterial lipopeptides is mediated by toll-like receptor 2, and is characterised by DC maturation and the enhanced capacity to stimulate of T cells. We report here that bovine DC are also induced to mature following lipopeptide stimulation. Exposure of DC to the model lipopeptide Pam3CSK4 was associated with increased expression of MHC, costimulatory molecules, and enhanced secretion of IL-12 and TNFalpha. Lipopeptide-matured DC were superior in their ability to induce T cell activation and IFNgamma secretion. In contrast, exposure of MPhi to lipopeptides induced down-regulation of MHC expression and much lower increases in IL-12 secretion. A lipopeptide derived from the sequence of a relevant mycobacterial lipoprotein, MPB83, also influenced bovine DC by stimulating increases in IL-12 and TNFalpha secretion. These different changes in bovine DC and MPhi may have important implications for immune responses induced following bacterial infection with uptake of microbes by DC resulting in potentiation of their immunostimulatory capacity and uptake by MPhi having a much less marked effect on immune responses.

Recognition of mycobacterial epitopes by T cells across mammalian species and use of a program that predicts human HLA-DR binding peptides to predict bovine epitopes.

Bioinformatics tools have the potential to accelerate research into the design of vaccines and diagnostic tests by exploiting genome sequences. The aim of this study was to assess whether in silico analysis could be combined with in vitro screening methods to rapidly identify peptides that are immunogenic during Mycobacterium bovis infection of cattle. In the first instance the M. bovis-derived protein ESAT-6 was used as a model antigen to describe peptides containing T-cell epitopes that were frequently recognized across mammalian species, including natural hosts for tuberculosis (humans and cattle) and small-animal models of tuberculosis (mice and guinea pigs). Having demonstrated that some peptides could be recognized by T cells from a number of M. bovis-infected hosts, we tested whether a virtual-matrix-based human prediction program (ProPred) could identify peptides that were recognized by T cells from M. bovis-infected cattle. In this study, 73% of the experimentally defined peptides from 10 M. bovis antigens that were recognized by bovine T cells contained motifs predicted by ProPred. Finally, in validating this observation, we showed that three of five peptides from the mycobacterial antigen Rv3019c that were predicted to contain HLA-DR-restricted epitopes were recognized by T cells from M. bovis-infected cattle. The results obtained in this study support the approach of using bioinformatics to increase the efficiency of epitope screening and selection.

The MPB83 antigen from Mycobacterium bovis contains O-linked mannose and (1-->3)-mannobiose moieties.

Mycobacterium tuberculosis and Mycobacterium bovis, the causative agents of human and bovine tuberculosis, have been reported to express a range of surface and secreted glycoproteins, although only one of these has been subjected to detailed structural analysis. We describe the use of a genetic system, in conjunction with lectin binding, to characterize the points of attachment of carbohydrate moieties to the polypeptide backbone of a second mycobacterial glycoprotein, antigen MPB83 from M. bovis. Biochemical and structural analysis of the native MPB83 protein and derived peptides demonstrated the presence of 3 mannose units attached to two threonine residues. Mannose residues were joined by a (1 --> 3) linkage, in contrast to the (1 --> 2) linkage previously observed in antigen MPT32 from M. tuberculosis and the (1 --> 2) and (1 --> 6) linkages in other mycobacterial glycolipids and polysaccharides. The identification of glycosylated antigens within the M. tuberculosis complex raises the possibility that the carbohydrate moiety of these glycoproteins might be involved in pathogenesis, either by interaction with mannose receptors on host cells, or as targets or modulators of the cell-mediated immune response. Given such a possibility characterization of mycobacterial glycoproteins is a step toward understanding their functional role and elucidating the mechanisms of mycobacterial glycosylation.