Chlamydia trachomatis is an obligate intracellular bacterium that alternates between two metabolically different developmental forms. We performed fluorescence lifetime imaging (FLIM) of the metabolic coenzymes, reduced nicotinamide adenine dinucleotides [NAD(P)H], by two-photon microscopy for separate analysis of host and pathogen metabolism during intracellular chlamydial infections. NAD(P)H autofluorescence was detected inside the chlamydial inclusion and showed enhanced signal intensity on the inclusion membrane as demonstrated by the co-localization with the 14-3-3β host cell protein. An increase of the fluorescence lifetime of protein-bound NAD(P)H [τ2-NAD(P)H] inside the chlamydial inclusion strongly correlated with enhanced metabolic activity of chlamydial reticulate bodies during the mid-phase of infection. Inhibition of host cell metabolism that resulted in aberrant intracellular chlamydial inclusion morphology completely abrogated the τ2-NAD(P)H increase inside the chlamydial inclusion. τ2-NAD(P)H also decreased inside chlamydial inclusions when the cells were treated with IFNγ reflecting the reduced metabolism of persistent chlamydiae. Furthermore, a significant increase in τ2-NAD(P)H and a decrease in the relative amount of free NAD(P)H inside the host cell nucleus indicated cellular starvation during intracellular chlamydial infection. Using FLIM analysis by two-photon microscopy we could visualize for the first time metabolic pathogen-host interactions during intracellular Chlamydia trachomatis infections with high spatial and temporal resolution in living cells. Our findings suggest that intracellular chlamydial metabolism is directly linked to cellular NAD(P)H signaling pathways that are involved in host cell survival and longevity. C. trachomatis infections is arduous and has not been comprehensively realized so far. A more detailed understanding about the metabolic activity and needs of C. trachomatis and its specific interactions with the host cell would be the basis for the development of novel treatment strategies. We therefore applied fluorescence lifetime imaging (FLIM) of the metabolic coenzymes NAD(P)H using two-photon microscopy to directly visualize metabolic changes of host cells and pathogens in living cells. NAD(P)H fluorescence was detected both on the chlamydial inclusion membrane and inside the inclusion. Interestingly, changes in chlamydial growth and progeny induced by glucose starvation and IFNγ treatment were directly linked to significant changes of the NAD(P)H fluorescence lifetimes inside the inclusions. Furthermore, measurement of the NAD(P)H fluorescence lifetime in the host cell nucleus revealed that infected cells were programmed for starvation during the metabolically active phase of intracellular chlamydial growth. Our findings highlight for the first time a direct interaction between host and pathogen metabolism in intracellular bacterial infections that exceeds sole competition for nutrients. In conclusion, fluorescence lifetime imaging of NAD(P)H by two-photon microscopy enables real-time analysis of metabolic host-pathogen interactions in intracellular infections with high spatial and temporal resolution.Separate analysis of host and pathogen metabolic changes in intracellular  Chlamydia trachomatis (C. trachomatis) has two metabolically different developmental forms, which ensure its infectivity and replication. The infectious form, the elementary body, enters the host cell and differentiates into a metabolically active form, the reticulate body. Chlamydial reticulate bodies grow within the host cell in an intracellular membrane-bound compartment called the chlamydial inclusion. Within 24 hours post infection (hpi), numbers and size of reticulate bodies are maximized and chlamydiae start to re-differentiate to infectious elementary bodies. Cell lysis and release of the elementary bodies occur at around 48 hpi The obligate intracellular bacterium C. trachomatis is a sensitive marker organism for host cell metabolic changes because it strongly depends on ATP and metabolites generated by the host. Although an ADP/ATP transporter has been found C. trachomatis not only uses host cell ATP, but also is capable to produce its own energy C. trachomatis are often truncated. Thus, C. trachomatis might directly import the substrates required to compensate for the incomplete metabolic pathways C. trachomatis according to sequence homology searches C. trachomatis after extracellular incubation The lack of suitable methods to investigate chlamydial metabolism separately from host cell metabolism has hindered scientific progress in studying host and pathogen metabolic interactions. Current knowledge on chlamydial metabolism is restricted to micro-array and RT-PCR analyses about the expression of metabolic genes during different intracellular developmental stages, the characterization of recombinant chlamydial metabolic enzymes, and the biochemical analysis of infected cells 2-NAD(P)H] is longer than that of free NAD(P)H [τ1-NAD(P)H] 2-NAD(P)H and the ratio of free to protein-bound NAD(P)H are established markers to sensor metabolic activity of eukaryotic cells and have been used to monitor changes in host cell glycolysis or oxidative phosphorylation 2-NAD(P)H while the inhibition of oxidative phosphorylation decreases τ2-NAD(P)H. The shift from oxidative phosphorylation to glycolysis has been made responsible for the decrease of τ2-NAD(P)H during cancer development Fluorescence lifetime imaging (FLIM) of NAD(P)H using two-photon laser scanning microscopy has been used to study cellular metabolism in living cells by utilizing the autofluorescence properties of the reduced form of the metabolic cofactor, NAD [NAD(H)] and its phosphorylated form, NADP [NAD(P)H] 2-NAD(P)H values and determining the ratio of free to protein-bound NAD(P)H in C.trachomatis-infected cells, we observed significant changes both in host cell and chlamydial metabolism during the intracellular developmental cycle.We applied two-photon FLIM of NAD(P)H to discriminate and thoroughly characterize chlamydial and host cell metabolism. By measuring τC. trachomatis-infected cells a new compartment is formed, the chlamydial inclusion which also showed a strong autofluorescence signal (2-NAD(P)H that indicates heterogeneity of NAD(P)H protein binding in each cellular compartment (2-NAD(P)H in the cytosol, nucleus, mitochondria and chlamydial inclusion were significantly different . The mean lifetime values are listed in 2-NAD(P)H was significantly increased inside the chlamydial inclusion at 24 hpi compared to all the other cellular compartments H in the cells . To confe signal . The spee signal . The chle signal . The selpartment . We coulartments .C. trachomatis-infected cells, high intensity signals only partially co-localized with TMRE. Most of the high intensity fluorescence which was found around the chlamydial inclusion did not co-localize with host cell mitochondria that were labeled with TMRE (C. trachomatis-infected cells (C. trachomatis-infected cells (NAD(P)H fluorescence showed an increased intensity surrounding the chlamydial inclusion. To test whether the high intensity signal originated from the host cell mitochondria, we co-incubated the cells with a mitochondria marker, tetramethylrhodamine-ethyl-ester (TMRE). TMRE showed complete co-localization with the high intensity NAD(P)H fluorescence in non-infected HEp-2 cells  and S3A.ith TMRE  and S3B.ed cells . Overexped cells .2-NAD(P)H inside the chlamydial inclusion are an indicator of chlamydial metabolism, we followed the alterations of τ2-NAD(P)H during the developmental cycle of C. trachomatis (2-NAD(P)H between 12 and 4 hpi. By contrast, τ2-NAD(P)H did not change further during the late phase of the infection (48 hpi) when infectious elementary bodies were formed (2-NAD(P)H was more heterogeneous at 48 hpi (1/a2 ratio) inside the chlamydial inclusion significantly decreased between 12 and 24 hpi (2-NAD(P)H was also observed in inclusions from early C. pneumoniae infected cells, followed by a decline in τ2-NAD(P)H during later time points of the infection H characterizes the metabolic activity of C. trachomatis, we monitored τ2-NAD(P)H changes inside the chlamydial inclusion during inhibition of host cell metabolism. We used glucose starvation to inhibit host cell glycolysis and antimycin A to inhibit the mitochondrial complex III and subsequent oxidative phosphorylation. Both inhibitors decreased host cell ATP levels (C. trachomatis was monitored by immunofluorescence staining and electron microscopic (EM) imaging of chlamydial inclusions (C. trachomatis progeny were measured by recovery assays (2-NAD(P)H inside the C. trachomatis inclusions when the host cells were treated with the metabolic inhibitors, as an indicator for decreased metabolism of C. trachomatis (2-NAD(P)H values was directly correlated to the decreased recovery rate of infectious C. trachomatis.To further prove that τP levels . The intclusions . Sizes oy assays . Both glchomatis  and S7B.C. trachomatis persistence that is defined as a viable but non-cultivable developmental stage. Persistent C. trachomatis are characterized by enlarged reticulate bodies in morphological aberrant inclusions 2-NAD(P)H values inside the chlamydial inclusions in IFNγ-treated cells 24 hpi. The mean values of τ2-NAD(P)H (2-NAD(P)H was also largely reduced resulting in an increase of the a1/a2 ratio (Interferon-γ (IFNγ) is known to induce -NAD(P)H  and the -NAD(P)H  were deca2 ratio .C. trachomatis-infection, FLIM analysis of cellular NAD(P)H was impeded by the large sizes of the chlamydial inclusions filling out almost the whole cytosol of the cells. As free NAD(P)H is diffusible, cytosolic levels are reflected in the cell nucleus. To characterize host cell metabolism during later stages of the infection, we measured the ratio of free to protein-bound NAD(P)H (a1/a2) and the values of τ2-NAD(P)H in the nucleus. The ratio of a1/a2 was decreased and τ2-NAD(P)H was increased in the nucleus of C. trachomatis-infected cells at 24 hpi H fluorescence around the chlamydial inclusion which was not of mitochondrial origin. Previous findings that the C. trachomatis-inclusion does not co-localize with the mitochondria of the host cells were confirmed chlamydiaechlamydiae also harbour a transporter rather than a biosynthetic pathway because of its smaller genome size.First we studied the compartmentalized distribution of NAD(P)H fluorescence in et al. that reticulate bodies first occupy the juxta-membrane space during the formation of the chlamydial inclusion and later move to the centre for differentiation Our data on the distribution of NAD(P)H fluorescence within the chlamydial inclusion further supports the model from Wilson 2-NAD(P)H values inside the chlamydial inclusion define a novel, metabolically distinct cellular compartment within infected cells. Comparison of the classical NAD(P) binding domains in humans and C. trachomatis using the SUPERFAMILY database search (http://supfam.cs.bris.ac.uk/SUPERFAMILY_1.73/) C. trachomatis (L2b/UCH-1/proctitis). Our measurements of τ2-NAD(P)H values during the developmental cycle indicate that NAD(P)H within C. trachomatis inclusion binds to different proteins as its metabolism changes. In addition, our results indicate that τ2-NAD(P)H strongly correlates with the metabolic activity of chlamydial reticulate bodies but not with the inclusion size. The increase in τ2-NAD(P)H in the mid- phase of infection was accompanied by a decrease in the relative amount of free NAD(P)H, which might indicate the presence of an oxidative type of energy metabolism within the chlamydial inclusion 2-NAD(P)H were measured in C. pneumoniae inclusions. The longer developmental cycle of the bacteria allowed also monitoring the decrease in metabolism towards the end of the infection cycle. However the absolute changes that we could measure were also less pronounced.The significantly increased τC. trachomatis2-NADP(H) and the ratio of a1/a2 in the persistent chlamydial inclusions we could prove that the metabolism of the chlamydial reticulate bodies are largely reduced. In addition, we could visualize the enlarged reticulate bodies inside the inclusions.IFNγ induces persistence of 2-NAD(P)H values in the chlamydial inclusions. Therefore, τ2-NAD(P)H values predicted metabolic activity of growing and differentiating reticulate bodies that resulted in infectious chlamydial progeny.Reduction of host cell ATP synthesis by inhibition of glycolysis or oxidative phosphorylation induced aberrant chlamydial inclusions and decreased τ2-NAD(P)H changes in the nuclei of C. trachomatis-infected cells were consistent with those found in 2FDG treated cells in which cellular glycolytic flux is inhibited and cellular starvation is induced within 24 hours Looking more in detail on the ratio of free to protein-bound NAD(P)H in the host cell nucleus, we could also show that intracellular chlamydial infection programmed the cell for starvation. Free NAD(P)H, which is approximately 80% of the total amount of NAD(P)H, represents the freely diffusing NAD(P)H molecules as described by measuring fluorescence lifetime of NADH in solution C. trachomatis inclusions in the first 12 hours of infection, a reliable analysis of the NAD(P)H fluorescence in the early phase of infection could not be performed.A limitation of NADP(H) autofluorescence imaging is that conversion of NAD(P)H fluorescence intensity values to absolute concentration values is not straightforward because the different quantum yields of free and protein-bound NAD(P)H have to be calculated C. trachomatis during the intracellular growth phase is needed to conceive novel therapeutic strategies to target the pathogen in its intracellular growth phase without affecting the host. Fluorescence lifetime imaging using two-photon microscopy bares new insights into the crosstalk between host and pathogen metabolism and suggests C. trachomatis-induced changes in sub-cellular NAD(P)H contents to directly interfere with nuclear NAD(P)H signaling pathways that are involved in cellular survival and longevity. In the process of understanding on how intracellular pathogens interfere with host cell metabolism, metabolic profiling of infected cells by FLIM of NAD(P)H will be an invaluable tool that complements established large scale genomic and proteomic approaches.A more detailed understanding about the metabolic activity and needs of 5 HEp-2 cells per dish were cultured in 50 mm culture dishes. For recovery assays, 3×105 HEp-2 cells per well were cultured in 6-well culture plates. Cells were allowed to adhere for 24 hours prior to infection with C. trachomatis or C. pneumoniae. Cells were grown at 37°C, in 5% CO2 humidified air. C. trachomatis strain L2 (ATCC VR-902B) and C. pneumoniae CWL029 strain (ATCC VR-1310) were purified on discontinuous density gradients. HEp-2 cells were infected with 1 inclusion forming unit (IFU) C. trachomatis per cell. HEp-2 cells were infected with 4 IFU C. pneumoniae per cell in the presence of 1 µg/ml cycloheximide by using centrifugation.HEp-2 epithelial cells (ATCC CCL-23) were cultured in Dulbecco's modified Eagle's Medium (DMEM) with glucose (4.5 g/l) , or for glucose starvation studies, in glucose free DMEM  supplemented with 10% heat-inactivated fetal bovine serum (FBS), 4 mM L-glutamine, 110 mg/l sodium-pyruvate, 10 mg/ml gentamicin and 30 mM Hepes. For two-photon microscopy, 5×10C. trachomatis or C. pneumoniae as described above. Cover glasses were examined in a MiniCeM chamber for microscopy  fitted to a heated stage, which enabled live cell imaging. The two-photon microscope  was equipped with a Chroma 640DCSPXR dichroic mirror  and a 40×/1.3 Plan-Apochromat oil-immersion objective . A tunable infrared titanium-sapphire femtosecond-laser  was used as an excitation source at 730 nm excitation for FLIM of NAD(P)H. Residual excitation light was blocked from the FLIM detector by a blue emission filter . FLIM data were collected by a time-correlated single-photon counting (TCSPC) system . Single photon counting was done for 49.7 seconds per image. Fluorescence lifetimes were analyzed using the SPCImage software version: 2.9.5.2996 (Becker & Hickl). The average power for the cell imaging experiments was 12 mW, and the scan area for each image plane was 110×110 µm2 corresponding to 256×256 pixels. Photon count rates at the beginning and end of image acquisition were monitored to ensure that photo-bleaching did not occur. For the image analysis, the ROI inside the chlamydial inclusion was selected. The ROIs contained 42 to 1147 pixels depending on the chlamydial inclusion size. The lifetimes of 5×5 pixels in the ROI were averaged before. The lifetime decay curves were fit to a double exponential decay model, in which the fast decaying component corresponds to free NAD(P)H [τ1-NAD(P)H] and the slow decaying component corresponds to protein-bound NAD(P)H [τ2-NAD(P)H[ 2-NAD(P)H of all pixels inside the ROIs were calculated. ROIs from three cells per microscopy field were analyzed from six different microscopy fields per chamber on three independent measurement days [n = 54 (3×6×3)]. To compare τ2-NAD(P)H of different infection times and metabolic states, values were normalized to the average τ2-NAD(P)H 24 hpi (100%) for each experimental day. For fluorescence intensity analysis of FLIM pictures, photon counts values of selected ROIs inside the chlamydial inclusion were exported to Excel from SPCImage analysis software. Values of each pixel inside the ROIs from three different cells were used to create a histogram of fluorescence intensity distribution.For two-photon microscopic studies, HEp-2 cells were grown on cover glass in 50 mm culture dishes and infected with Fluorescence light was collected in four emission channels simultaneously using a set of dichroic mirrors and four photomultiplier tubes (Hamamatsu R1294A and R1295A). Fluorescence emission was spectrally distributed as follows: channel 1: 380–450 nm, channel 2: 450–500 nm, channel 3: 500–580 nm, channel 4: 580–680 nm. Residual excitation light was blocked by a two-photon emission filter . Fluorescence intensities in each emission channels were analyzed in medium and in ROIs in host cell mitochondria and chlamydial inclusion by ImageJ. Background fluorescence intensity was recorded in a dark measurement without excitation light and was subtracted from the autofluorescence intensity values. The peak of emission in channel 2 (emission between 450–500 nm) at 730 nm excitation by two-photon microscopy corresponds to the peak of NAD(P)H fluorescence spectrum C. trachomatis infection using 10 virus particles per cell. The plasmid encoding the carboxy terminally GFP-tagged 14-3-3β protein (RG215940) was purchased from Origene . Transfection was performed 24 hours before C. trachomatis infection using 2 µl/ ml Fugene6 transfection reagent (Roche) and 1 µg / ml DNA according to the manufacturer's instruction.HEp-2 cells were transduced with CellLight Golgi-GFP BacMam 2.0 or CellLight Lysosomes-GFP BacMam 2.0 (Invitrogen). Transduction was performed simultaneously with GFP was excited at 840 nm by the two-photon microscope described above. Cellular autofluorescence was minimal at 840 nm when low excitation power was used. Two-photon excitation of GFP fluorescence at 730 nm provided minimal absorption when GFP overexpression was at a low level. To analyze co-localization, intensity images were exported to the Keyence BZ9000 Analysis software  and line intensity profiles of the fluorescence were calculated.Chlamydial inclusion sizes were analyzed by measuring the area of inclusions after exporting the grey-scale FLIM intensity images from SPCImage software to ImageJ . 54 inclusions were analyzed from three independent experiments.C. trachomatis infection.To induce persistence, HEp-2 cells were treated with 10 units/ml IFNγ for 24 hours prior to and during C. trachomatis elementary bodies after intracellular development, titration experiments were performed. Infected HEp-2 cells (24 hpi) were mechanically detached with a cell scraper and re-suspended in fresh growth medium. Serial dilutions of suspension were inoculated in confluent cycloheximide-treated HEp-2 cell monolayers with centrifugation-assisted inoculation. Development of chlamydial inclusions after 30 hours was analyzed on methanol-fixed slides using FITC-labeled monoclonal chlamydial-LPS antibodies . Chlamydial recovery was calculated as IFU/µl by observation of 10 microscopy fields (40×magnification).To determine the burden of infectious C. trachomatis or were left un-infected. After 24 hours of infections, FLIM measurements, recovery assays, ATP measurements and immunofluorescence stainings were performed.To inhibit host cell glycolysis, glucose starvation or 2FDG (Sigma) treatment were used. To inhibit host cell oxidative phosphorylation, antimycin A (Sigma) treatment was used. HEp-2 cells were allowed to adhere for 24 hours. For glucose starvation, media was changed to media containing no glucose. For 2FDG and antimycin A treatment, media was changed to media containing 5 mM 2FDG or 3 nM antimycin A respectively. 15 minutes after the media was changed, cells were infected with C. trachomatis. After 24 hours of infection, cells were fixed with methanol and chlamydial inclusions were stained with FITC-labelled monoclonal chlamydial-LPS antibodies (Dako). For better visualization cells were counterstained with 0.1% Evans-blue.Growth media was changed to media containing no glucose or containing antimycin A (3 nM) prior to infection with ATP was measured using a luminometric ATP assay kit  and a microplate reader . HEp-2 cells were grown in 96-well plates (2000 cells/ well) and treated with the metabolic inhibitors as described above. ATP assays were performed according to the manufacturer's instructions.Cells were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 1 hour. Post-fixation was performed with 1% OsO4 in O.1 M cacodylate buffer for 2 hours. Samples were dehydrated with graded ethanol series and embedded in araldite . Ultrathin sections were stained with uranyl acetate and lead citrate and were examined with a Philips EM 400 transmission electron microscope.For all comparisons, we used nonparametric models for longitudinal data as described previously 1433B_HUMAN (YWHAB): P31946.Figure S1Spectral characterization of autofluorescence signals at 730 nm excitation. (A) Spectral characterization of autofluorescence signals of non-infected HEp-2 cells (upper panels) and of C. trachomatis-infected cells at 24 hpi (lower panels) shows typical NAD(P)H fluorescence . (B) Quantification of fluorescence intensity of selected ROIs (white squares) in host cell mitochondria, cytosol, nucleus and chlamydial inclusion at 24 hpi . Fluorescence intensity values were normalized to the fluorescence intensity of the media in each emission channels. There is no shift in the peak emission wavelengths of autofluorescence originating from the chlamydial inclusion compared to host cell mitochondria. The peak of emission between 450–500 nm at 730 nm excitation by two-photon microscopy corresponds to the peak of the NAD(P)H fluorescence spectrum.(TIF)Click here for additional data file.Figure S22-NAD(P)H.Autofluorescence intensity and frequency distribution of τ (A) Autofluorescence intensities were used to define different cellular compartments (upper panels) and ROIs were transferred to color-coded images of τ2-NAD(P)H (lower panels) of a non-infected and a C. trachomatis-infected cell at 24 hpi . The line in the C. trachomatis-infected cell (upper right) marks the area that was used for analysis of fluorescence intensity in C. trachomatis-infected cell. The fluorescence intensity is different in host cell mitochondria, nucleus and inside the chlamydial inclusion indicating the different concentration of NAD(P)H in these cellular compartments. The different intensity signal enables the separation of these cellular compartments . (C) Histogram of τ2-NAD(P)H in the cytosolic, mitochondrial and nuclear compartments of non-infected cells and in the chlamydial inclusion of infected cells at 24 hpi (n = 54 from three independent experiments).(TIF)Click here for additional data file.Figure S3Co-localization analysis of NAD(P)H fluorescence with the mitochondria marker, TMRE. (A) Intensity profile (middle panel) of the line indicated in the overlay picture (upper panel) and in the magnified picture (lower panel) show complete co-localization of TMRE and NAD(P)H fluorescence in non-infected HEp-2 cells. (B) Intensity profile (middle panel) of the line indicated in the overlay picture (upper panel) and in the magnified picture (lower panel) show co-localization of TMRE and NAD(P)H fluorescence in the mitochondria but not in the chlamydial inclusion and on the inclusion membrane in C. trachomatis-infected HEp-2 cell at 24 hpi.(TIF)Click here for additional data file.Figure S4FLIM analysis of C. trachomatis metabolism. (A) The scatter plot shows the homogenous distribution of the average τ2-NAD(P)H values in C. trachomatis inclusions at 24 hpi and the heterogeneous distribution at 48 hpi (n = 54 from three independent experiments). (B) Quantitative analysis of the ratio of free to protein-bound NAD(P)H (a1/a2) inside the C. trachomatis inclusion of NAD(P)H FLIM images after the indicated time points of infection . Detailed results of statistical analysis are shown in (TIF)Click here for additional data file.Figure S5NAD(P)H autofluorescence intensity in the chlamydial inclusion 18 hpi, 24 hpi and 48 hpi. (A) Pseudo-colour images of NAD(P)H fluorescence intensity measured by FLIM in C. trachomatis-infected cells 18 hpi, 24 hpi and 48 hpi . (B) Histogram of NAD(P)H autofluorescence distribution inside the chlamydial inclusion after 18 hours (n = 442 pixels), 24 hours (n = 1060 pixels) and 48 hours (n = 1561 pixels) of infection. (C) Enlarged images of representative cells used for NAD(P)H fluorescence intensity analysis .(TIF)Click here for additional data file.Figure S62-NAD(P)H inside Changes of τC. pneumoniae inclusions during the intracellular developmental cycle. (A) Changes of τ2-NAD(P)H inside the C. pneumoniae inclusion during the bacterial developmental cycle. HEp-2 cells were infected with C. pneumoniae for 36, 48, 60, 72, 84 and 96 hours. (B) Quantitative analysis of τ2-NAD(P)H inside the C. pneumoniae inclusion and of C. pneumoniae inclusion sizes (n = 36 (36 hpi); n = 32 (48 hpi); n = 42 (60 hpi); n = 27 (84 hpi); n = 18 (96 hpi) from four independent experiments; mean±SEM).(TIF)Click here for additional data file.Figure S72-NAD(P)H.Impact of host-cell metabolism inhibition on ATP levels and τ (A) Cellular ATP levels under glucose starvation and inhibition of oxidative phosphorylation by antimycin A. (B) Histogram of τ2-NAD(P)H in the C.trachomatis-inclusions under different metabolic perturbations.(TIF)Click here for additional data file.Table S1Quantitative analysis of NAD(P)H FLIM. Fluorescence lifetimes, relative amounts and fluorescence quantum yields of free and protein-bound NAD(P)H in the cytosol, mitochondria and nucleus of non-infected HEp-2 cells and in the C. trachomatis inclusion. τ1: fluorescence lifetime of free NAD(P)H, τ2: fluorescence lifetime of protein-bound NAD(P)H, a1: relative amount of free NAD(P)H, a2: relative amount of protein-bound NAD(P)H, q1: fluorescence quantum yield of free NAD(P)H, q2: fluorescence quantum yield of protein-bound NAD(P)H .(DOC)Click here for additional data file.Table S22-NAD(P)H in cytosol, mitochondria, nucleus of non-infected HEp-2 and in chlamydial inclusion at 24 hpi.Statistical analysis of τ The model included experimental days  and compartment  as independent factors and images per day (six) as well as cells per image (three) as repeated measures with all main effected and interactions. The dependent variable was τ2-NAD(P)H in nucleus. Compartment was tested overall as well as comparing inclusion with every other compartment.(DOC)Click here for additional data file.Table S32-NAD(P)H and a1/a2 in the chlamydial inclusion 12 hpi, 24 hpi and 48 hpi.Statistical analysis of τ The model included experimental days  and time point  as independent factors and images per day (six) as well as cells per image (three) as repeated measures with all main effected and interactions. The dependent variable were τ2-NAD(P)H (A) and a1/a2 (B). Differences in time points were tested.(DOC)Click here for additional data file.Table S42-NAD(P)H in the chlamydial inclusion under different metabolic inhibition conditions at 24 hpi.Statistical analysis of τ The model included experimental days  and treatment  as independent factors and images per day (six) as well as cells per image (three) as repeated measures with all main effected and interactions. The dependent variable was τ2-NAD(P)H.(DOC)Click here for additional data file.Table S52-NAD(P)H and a1/a2 in the host cell nucleus.Statistical analysis of τ The models included experimental days  and treatment  as independent factors and images per day (six) as well as cells per image (three) as repeated measures with all main effected and interactions. The dependent variables were τ2-NAD(P)H (A) and a1/a2 (B).(DOC)Click here for additional data file.


Paramyxoviridae family virion, we hypothesized that the inflammatory modulation observed in hMPV infected patients may be partly associated with the matrix protein (M-hMPV) response. By western blot analysis, we detected a soluble form of M-hMPV released from hMPV infected cell as well as from M-hMPV transfected HEK 293T cells suggesting that M-hMPV may be directly in contact with antigen presenting cells (APCs) during the course of infection. Moreover, flow cytometry and confocal microscopy allowed determining that M-hMPV was taken up by dendritic cells (moDCs) and macrophages inducing their activation. Furthermore, these moDCs enter into a maturation process inducing the secretion of a broad range of inflammatory cytokines when exposed to M-hMPV. Additionally, M-hMPV activated DCs were shown to stimulate IL-2 and IFN-γ production by allogeneic T lymphocytes. This M-hMPV-mediated activation and antigen presentation of APCs may in part explain the marked inflammatory immune response observed in pathology induced by hMPV in patients.Each year, during winter months, human Metapneumovirus (hMPV) is associated with epidemics of bronchiolitis resulting in the hospitalization of many infants. Bronchiolitis is an acute illness of the lower respiratory tract with a consequent inflammation of the bronchioles. The rapid onset of inflammation suggests the innate immune response may have a role to play in the pathogenesis of this hMPV infection. Since, the matrix protein is one of the most abundant proteins in the  Pneumoviridae subfamily of Paramyxoviridae, has been recognized as a leading cause of acute respiratory disease in children worldwide Human Metapneumovirus (hMPV), a member of the DCs are the most influential APC A characteristic of the paramyxovirus family is that they have a lipid envelope containing two membrane glycoproteins: an attachment glycoprotein (G) and a fusion protein (F). The nucleocapsid core contains the viral RNA genome as well as the nucleocapsid (N), phospho- (P), and large (L) proteins in vitro, human macrophages and DCs can be activated by the recombinant M-hMPV protein. Our findings provide new insights about the role of M-hMPV protein in the modulation of the immune response to virus and may provide information to support a better understanding of hMPV pathogenesis.In the present study, we show that the M-hMPV protein can be released in a soluble form and The hMPV strain stock NL/1/00 corresponding to supernatant and cells was obtained from Pr. A.D.M.E Osterhaus and Dr. J. Simon at ViroNovative B.V., Rotterdam, The Netherlands. Briefly, Vero cells were infected with the hMPV/NL/1/00 M-hMPV protein was detected from supernatant of hMPV infected Vero cells using positively charged magnetic beads obtained from Dr. R. Veyret, bioMérieux, Lyon, France Western blotting was performed by using 1 µg/mL 8D12F6 and 1D7A7 monoclonal antibodies directed against M-hMPV . The membrane was washed 10 min, three times in PBS-T and then incubated for 1 h at room temperature with 1 µg/mL goat anti-mouse immunoglobulin G horseradish peroxydase antibody (Promega). After three washes in PBS-T for 10 min each time, the membrane was developed by ECL-Western blotting detection reagents  according to the manufacturer's instructions and exposed to Kodak X-OMAT film for 45 min.E. coli and purified as described previously 5′-ATGGAGTCCTACCTAGTAGA-3′ using Thermoscript RT kit (Invitrogen)  followed by two complementary PCR assays using the taq polymerase high fidelity kit with primers 5′-ATGGAGTCCTACCTAGTAGA-3′ and 5′-TCTGGACTTCAAGACATATC-3′  and 5′-CTCTCTGCTAGCGAGTCCTA CCTAGTAGACACCTATCAAGG-3′C and 5′-CTCTCTGGATCCTTAGTGGT GATGGTGATGGTGAGAACCC CTCATTCTGGACCTCAAGAC ATATCT-3′, . These primers introduce a MRGS-6xHis tag at the C-terminal end. The NheI and BamHI (underlined) digested PCR products were ligated into the digested pET21-b expression vector (Novagen). E. coli BL21DE3 bacteria containing recombinant pET21-b/M-hMPV was grown until the OD600 reached 0.6 and induced with 0.4 mM isopropyl-beta-D-thiogalactopyranoside. After 3 h, bacteria cells were centrifuged and resuspended in a lysis buffer, disrupted by sonication (3×10 s–30 watts), followed by further centrifugation. M-hMPV protein was absent from supernatant and was then extracted from pelleted bacteria by a 6M urea buffer. This was then applied on Ni-NTA Agarose (Qiagen) for purification according to the manufacturer's instructions in endotoxin-free conditions. 6xHis-tagged recombinant M-hMPV protein was eluted from the column by a pH gradient. Analysis of eluted fraction was performed with Coomassie brilliant blue staining after electrophoresis on 12% SDS-polyacrylamide gel under reducing conditions.cDNA was obtained from a RNA sample extracted using the Nuclisens kit (bioMérieux) from nasal swab of children mono-infected by hMPV genotype A1 and hospitalized with bronchiolitis. The M-hMPV (nucleotides 2165–2929) was expressed in Mass spectrometry analysis was completed using MALDI-TOF in a voyager-DE-PRO  and only the M-hMPV protein was observed.The endotoxins concentration of the purified M-hMPV was determined by a kinetic chromogenic-QCL Bio Wittaker Limulus amoebocyte lysate assay at Laboratoire Marcel Mérieux  and was negative (<0.25 U endotoxins/mg of M-hMPV protein). The RNA contaminants detection was performed by RNA Array 6000 Pico kit from Agilent (Agilent Technologies) and by detection of RNA 16S with universal PCR Light Cycler Fast Start Master SYBR Green I. The protein M-hMPV preparation did not contain RNA contaminants. The M-hMPV protein was labeled by using Fluorescein-NHS (Pierce) and the preparation controlled for the absence of endotoxins. Free fluorescein was removed by dialysis and labeled protein was filtered at 22 µM and recovered in PBS/SDS 0.01%/Azide 0.9 g/L.BamHI restriction site. M-hMPV sequence was amplified from bacterial expression plasmid pET21b/M-hMPV by PCR using primer introducing BamHI restriction site: 5′-CTAGGATCCCACCATGGCTAGCGAGTCCTAC-3′ and 5′-CTAGGATCCTTATCTGGACTTCAAGACATATC-3′. phCMV/M-hMPV was transfected in HEK 293T cells 2 flask to a confluence of 60%. Transfection was performed using FuGENE HD (Roche) according to the manufacturer's instructions. Briefly, 10 µg of DNA was diluted in 1000 µL of serum-free DMEM, and 30 µL of FuGENE HD was added to the diluted DNA. After 15 min of incubation, the mixture was added to the cells and incubated for 6 h at 37°C. Then, 30 mL of DMEM supplemented with 10% FCS was added to the cells.The M-hMPV coding sequence was cloned into the vector phCMV in g, 4°C) to remove cellular debris. The clarified supernatant was loaded on a 20% sucrose cushion and centrifuged at 250 000 rpm at 4°C for 3 h in a Beckman SW41 rotor ultracentrifuge. After ultracentrifugation the upper phase containing soluble protein was harvested and the pellet was resuspended in 1 mL of PBS.48 h after transfection, the cells supernatant was harvested and centrifuged  wt/wt, loaded at the bottom of a tube, and layered with 8 mL 40% histodenz and 2 mL of 10% histodenz. Tubes were centrifuged for 20 h at 250 000 rpm in a Beckman SW41 rotor.6 cells/mL) were differentiated to immature DCs (iDCs) and macrophages (MDMs Macrophage-derived monocytes) in complete RPMI 1640 medium containing 2 mM glutamine, 10 mM HEPES, 40 ng/mL gentamicin  and 10% FCS (Biowest), supplemented with 40 ng/mL human recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF) plus 250 U/mL human recombinant IL-4 for iDCs or 40 ng/mL macrophage colony-stimulating factor (M-CSF) for MDMs  and incubated for 5 days at 37°C in 5% CO2.Human peripheral blood from healthy donors was obtained from the Etablissement Français du Sang . Mononuclear cells were isolated by density gradient centrifugation using Ficoll-Hypaque, and then centrifuged on a 50% Percoll solution (Amersham Biosciences). Two fractions were recovered: monocytes were purified from the light density fraction and T lymphocytes from the high density fraction. The purification of monocytes and lymphocytes were performed by immunomagnetic beads depletion  using a cocktail of monoclonal antibodies (mAb): anti-CD3, anti-CD16, anti-CD19, anti-CD56 and anti-glycophorin A for monocytes purification and anti-CD14, anti-CD16, anti-CD19, anti-CD56, and anti-glycophorin A for lymphocytes purification . Monocytes were >95% enriched as assessed by CD14 labeling and T lymphocytes >95% pure as assessed by CD3 labeling. Monocytes (106/mL) for 20 min on ice. Cells were then washed and recovered in cold FACS buffer. In competition experiments, 4.1 nM M-hMPV-Fluorescein was incubated with cells during a 20 min period on ice, washed twice by cold medium and an increased non-labeled concentration of M-hMPV was then added to the cells and incubated on ice for 20 min. Those cells were then washed and recovered in cold FACS buffer. To follow receptor-mediated endocytosis, cells were incubated on ice for 20 min with 1.3 nM Fluorescein-M-hMPV, washed twice at 4°C, and then incubated at 37°C for 5 to 30 min in complete RPMI medium. Internalization was stopped on ice with cold PBS containing 0.1% BSA and 0.05% NaN3. 2×105 cells of binding and internalization experiments were added per round 12 mm diameter coverslip (Marienfeld) coated with poly-l-lysine (Sigma) and incubated 1 h at 4°C for binding and at 37°C for internalization. Cells were fixed using 4% paraformaldehyde (PFA) for 10 min, rinsed with PBS and examined by confocal microscopy using a non inverted Axioplan2 LSM510 Zeiss, as well as the Zeiss LSM Image Browser software (Zeiss).The binding of Fluorescein-M-hMPV was performed on differentiated cells harvested on day 5 and resuspended in culture medium. Different concentrations of Fluorescein-M-hMPV were incubated with cells (10E. coli isotype 0111:B4) (Sigma) was used as a positive control of phenotype maturation and HCV-NS3 Helicase recombinant protein (HCV-NS3H) produced in the same conditions as the M-hMPV protein as a negative control.To determine its effect, the M-hMPV recombinant protein was added to the iDCs for 24 h. We tested the bacterial recombinant M-hMPV protein as well as different protein controls: the protein elution buffer, the protein treated by polymyxin B sulphate (10 µg/mL) (Sigma), and heated at 20 min at 100°C. Also, LPS (10 ng/mL)  using CELLQUEST software (Becton Dickinson) (10 000 events/test). FITC-conjugated anti-CD14, -HLA-DR, -CD80, and phycoerythrin (PE)-conjugated anti-CD1a, -CD86, -CD83 and -CD40 were used to phenotype the cells . An isotypic control was also used to set the gating for the cells.6 moDCs were pelleted, re-suspended in 100 µL of PBS containing 40 nM 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3) (Molecular Probes) and incubating 30 min at room temperature in the dark. After incubation, cells were washed twice in PBS and re-suspended in FACS buffer. The DiOC6(3) membrane potential-related fluorescence was analyzed by flow cytometry on a FACS Calibur.1×106 DiOC6(3) labelled cells prior analysis by flow cytometry.Quantification of apoptotic nuclei was also assessed by staining apoptotic nuclei with Propidium Iodide. Briefly, 25 µM of PI was added to the 1×10Supernatants from moDCs were assayed for cytokines IL-12p70, TNF, IL-10, IL-6, IL-1β, and IL-8 and supernatants from MDMs were assayed for chemokines MIP-1β, RANTES and TNF by Cytometric Bead Array . IFN-α and IFN-β were measured using ELISA kits .2, cells were collected, extensively washed, resuspended in RPMI 10% FCS and then co-cultured with 2×105 allogeneic T-cells in 200 µL of complete culture medium at 1∶5, 1∶10 or 1∶20 APC: responder T-cell ratio. After co-culture at 37°C in 5% CO2, supernatants were recovered and tested for an IL-2 (day 2), IL-5, IL-13, and IFN-γ (day 5) secretion assay by Cytometric Bead Array flex (BD Biosciences).Mixed lymphocyte reaction (MLR). Naive T-lymphocytes were purified from human peripheral blood as previously described in section “Generation of monocyte-derived DCs (moDCs) and monocyte-derived macrophages (MDMs)”. The cell assays were carried out in 96-well flat-bottom culture plates in triplicate for each condition. iDCs were treated for 24 h with LPS (positive control), HCV-NS3H protein (negative control), elution buffer and M-hMPV protein. After incubation at 37°C in 5% COWe hypothesized that M-hMPV can be released from hMPV infected cells. To confirm this hypothesis, we captured the protein from the supernatant of hMPV infected Vero cells. After ultracentrifugation to remove virus particles, anionic charge polymers beads were used to capture the free soluble M-hMPV  confirmiTo support the previous result, 293T cells were transiently transfected by the vector phCMV encoding M-hMPV. A soluble form of M-hMPV in the supernatant of transfected cells was detected and analyzed. As seen in in vitro by cultivating human monocytes with differentiation factors. Analysis of the moDCs phenotype showed that at day 5 of culture, these cells had little expression of CD14 but high levels of CD1a and intermediate levels of HLA-DR . After 24 h of contact with the M-hMPV protein, the phenotype of moDCs was analysed by flow cytometry and by measuring the expression of CD40, CD80, CD83 and CD86 surface molecules. As shown in In addition to the expression of surface co-stimulatory molecules, the DCs maturation process can lead to the release of soluble mediators, such as cytokines, which affect both humoral and T-cell-mediated immune responses. As shown in M-hMPV induced type I interferon response. The type I interferons (IFNs) play an essential role in the early antiviral cell response. With this in mind, we investigated the potential for M-hMPV to induce these molecules. The secretion of type I IFNs was quantified in MDMs supernatants obtained after 24 h of culture with M-hMPV. M-hMPV treated-MDMs produced only low levels of IFN-β , and simTo further study the maturation level and the functionality of the moDCs, we tested M-hMPV activated DC for their capacity to stimulate the allogeneic T-cells by performing a MLR. The mature moDCs treated with the M-hMPV protein were co-cultured with allogeneic T-cells for either two or five days. Supernatants were recovered and the production of IL-2 (day 2), IFN-γ, IL-5 and IL-13 (day 5) was analyzed. The allogeneic T-cells produced IFN-γ in a ratio- and dose-dependant manner . FurtherhMPV is a major cause of respiratory tract illness especially in young children and the need to develop new strategies to deal with such infection remains a priority. The matrix protein is one of the most abundant protein in the virion and its role in the establishment of the immune response is still unknown. The M-hMPV soluble protein was detected in the supernatant of both hMPV infected Vero cells and transiently M-hMPV transfected 293T cells. Because the M-hMPV release has been detected in the supernatant of cells presenting characteristic hMPV cytopathic effects, a hypothesis could be that a process of cell death may occur and allow the release of the matrix protein from infected cells. Further experiments are needed to characterize the release process.The release from the hMPV infected cells can be the result of a specific mechanism or of cell death. Regarding the transiently M-hMPV transfected 293T cells, most of the protein was released as a soluble form leaving a small proportion of the protein associated with the cell membranes. To our knowledge, this is the first time that a soluble viral matrix protein has been reported. The M-hMPV protein does not have a signal peptide and may not be able to enter the conventional secretion pathway The objective of this study was to determine the M protein effect in the immune response. To further study M-hMPV protein, an endotoxin free M-hMPV recombinant protein was produced in bacteria. In our model, M-hMPV protein demonstrated a dose-dependent binding to moDCs and MDMs along with an efficient and rapid internalization as demonstrated by confocal microscopy. Incubation of moDCs with M-hMPV also led to the up-regulation of co-stimulatory molecules CD40, CD80, CD83 and CD86 indicating that moDCs appeared to be activated and induced to mature. Furthermore, the M-hMPV treatment does not induce apoptosis of the moDCs that could explain the release of cytokines/chemokines. This observed maturation response was unlikely to be caused by LPS contamination as polymyxin B did not affect M-hMPV-induced phenotypic or functional maturation. Heating M-hMPV completely abolished its activation effects whereas LPS is inactivated by heating. Furthermore, M-hMPV protein did not contain detectable nucleic acid contaminants (data not shown) that could activate a Toll-like receptor pathway.in vitro model, we observed that moDCs and MDMs treated with M-hMPV were induced to produce cytokines and chemokines associated with their maturation and activation. M-hMPV protein induced a dose-dependent increase of TNF and IL-12p70 production, essential inflammatory cytokines that have been associated with immune responses including Th1-type et al.et al. firstly reported an outbreak of hMPV infection in a long-term facility care and presented histopathological findings associated with a fatal pneumonia case in an 89-year-old woman In our The complete maturation of M-hMPV treated-moDCs was demonstrated by their ability to stimulate IFN-γ synthesis by allogeneic T cells indicating that M-hMPV protein is a predominant Th1 promoting innate stimulus. This IFN production is controlled by cytokines secreted by APCs, particularly by IL-12. IFN-γ up-regulates the expression of class I MHC molecules to the cells, which is important for the immune host response as it increases the recognition of pathogens peptides by cytotoxic T cell and induces the cell mediated immunity et al.et al.et al.IFN. The hMPV glycoprotein G has been identified as an important virulence factor that interacts with and inhibits the activation of RIG-I, an RNA helicase which is involved in the signaling cascade leading to NF-κB, IRF activation and IFN production. The glycoprotein G of hMPV appears to inhibit the production of antiviral molecules like the IFNs. Depending of the cell type, hMPV induces or not a production of type I IFN. It may be of some interest to determine if the M-hMPV protein can act like the glycoprotein G and inhibits the IFNs production by interacting with a sensor of viral infection in DCs.The induction of type I IFN molecules is crucial in the establishment of the innate immune response to fight a viral infection et al.et al.et al.Paramyxoviridae family that cause indistinguishable clinical symptoms. However, data discussed previously, demonstrate a difference in the immune response. It is interesting that, unlike RSV, hMPV did not stimulate the production of inflammatory cytokines, hMPV-infected DC were able to stimulate T cells in vitro may explain the paradigm of the results obtained by Guerrero-Plata et al. and Tan et al. in their studies.Tan The primary defense barriers against pathogens, such as viral particles or proteins of pathogens, are built up by epithelial surfaces in contact with APCs. We hypothesized that hMPV infection in pulmonary epithelial cells may lead to the release of M-hMPV protein stimulating the APCs resident in the respiratory tract. M-hMPV could then selectively and efficiently be processed by APCs. Moreover, M-hMPV induces their maturation, promotes their capacity to stimulate T lymphocytes and finally elicits a predominant Th1 pattern of response.In conclusion, we demonstrate for the first time that M-hMPV protein can be released in a soluble form and that it can interact with human moDCs and MDMs inducing a response. These M-hMPV/APCs interactions were characterized by (1) the binding and internalization of M-hMPV to human APCs in a dose-dependent manner; (2) the up-regulation of moDCs co-stimulatory molecules CD40, CD80, CD83 and CD86; (3) the secretion of inflammatory mediators IL-8, IL-6, TNF, IL-12p70, IL-1β and IL-10; and (4) the stimulation of T cells by moDCs activated by M-hMPV inducing a predominant Th1 polarization and demonstrating an efficient M-hMPV innate stimulus. These finding suggest that M-hMPV plays a central role in the inflammatory response initiated by the host innate immune system and is highly intertwined in the mechanisms of respiratory pathogenesis.


Leishmania (L.) tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases ) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB-CTE-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB-CTE-recombinant L. tarentolae as a safe live vaccine candidate against VL.Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans  Leishmania tarentolae is considered as nonpathogenic to humans. In our previous work, a recombinant L. tarentolae strain expressing the amastigote-specific L. donovani A2 antigen as a vaccine candidate elicited protection against L. infantum challenge in mice. Furthermore, combinations of CPA/CPB cysteine proteinases were more protective against visceral and cutaneous Leishmania infections than the individual forms. Herein, we used DNA/Live and Live/Live prime-boost vaccination strategies against visceral leishmaniasis in BALB/c mice consisting of the A2-CPA-CPB-CTE tri-fusion genes formulated with cationic solid lipid nanoparticles and a recombinant L. tarentolae expressing the tri-fusion. Assessments of cytokine production, humoral responses, parasite burden and histopathological studies support that the recombinant L. tarentolae A2-CPA-CPB-CTE candidate vaccine elicits a protective response against visceral leishmaniasis in mice and represents an important step forward in the development of new vaccine combinations against Leishmania infections.Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and has emerged as an opportunistic infection in HIV-1 infected patients in many parts of the world. Drug-resistant forms have developed so emergence and increased the need for advanced preventive strategies. Using live avirulent organisms as a vaccine has been proven to be more effective than other regimens. The lizard protozoan parasite  Leishmania species that ranges from self-limiting cutaneous leishmaniasis to fatal visceral leishmaniasis (VL) and is endemic in 88 tropical and subtropical countries L. donovani complex with a wide and growing prevalence and incidence (http://www.who.int/en/) L. infantum is responsible for VL in the Mediterranean basin, extending to several Middle East and Asian countries Leishmania included recombinant proteins, polyproteins, DNA vaccines, liposomal formulation, and dendritic cell vaccine delivery systems in vitro culturing Leishmania genes encoding virulence factors or enzymes responsible for their synthesis and genes essential for intracellular survival have been reported Leishmania species, an approach comparable to the use of BCG as a vaccine against Mycobacterium tuberculosis infections.Leishmaniasis is a vector-borne disease caused by different Leishmania, the lizard protozoan parasite L. tarentolae has never been found associated with any form of human leishmaniasis and is therefore considered nonpathogenic L. tarentolae is capable of infecting mammalian cells and transforming into amastigote-like forms, it is not however able to persist long enough within macrophages L. tarentolae as a vaccine vector to deliver specific Leishmania antigens mimicking live infection has also been explored. In a previous study, the L. donovani amastigote-specific A2 antigen was expressed in L. tarentolae, which lacks this protein L. infantum challenge and was associated with the production of high levels of IFN-γ production Among different species of L. major and L. infantumL. infantum infection and therefore is not protective as a vaccine candidate -CTE is more protective against visceral and cutaneous leishmanial infections than the individual forms It has been reported that sera from either cured or active cases of cutaneous and visceral leishmaniasis patients recognize the recombinant cysteine proteinases CPA (rCPA) and CPB (rCPB) of -CTE tri-gene fusion as a DNA vaccine formulated with cSLN and also a recombinant L. tarentolae expressing the tri-gene fusion as a live vaccination strategy against visceral leishmaniasis in two-modalities, namely DNA/Live and Live/Live vaccination in BALB/c mice. We demonstrate that prime-boost strategies harboring recombinant L. tarentolae-based vaccines represent a promising immunization approach against Leishmania infections.Here, we use the A2-CPA-CPBAll solutions were prepared using MilliQ ultrapure  and apyrogenic water to avoid surface-active impurities. Cetyl palmitate, Tween 80 and cholesterol were purchased from Merck . G418, N-[1- propyl]-N,N,Ntrimethylammonium chloride (DOTAP), Sodium dodecyl sulfate (SDS) were purchased from Sigma-Aldrich . The materials applied for PCR, enzymatic digestion and agarose gel electrophoresis were acquired from Roche Applied Sciences . Cell culture reagents including M199 medium, HEPES, L-glutamine, adenosine, hemin, gentamicin, RPMI and Schneider were sourced from Sigma  and Gibco , respectively. Fetal Calf Sera (FCS) was purchased from .A2 gene (with Kozak sequence) was digested from pUC57 vector  with EcoRI and HindIII restriction sites. After sequence confirmation, the A2 fragment was subcloned into the EcoRI and HindIII sites of vector pGEM7zf(+) (Promega). The CPA fragment was amplified from pGEM-CPA using Taq DNA Polymerase  and the following primers:  including HindIII restriction site (underlined); and  including NheI restriction site (underlined). Then, the PCR-amplified CPA gene was cloned into HindIII and NheI sites of pAT153 vector (Boca Scientific). The CPB-CTE fragment was amplified from pGEM-CPB using Taq DNA Polymerase  and the following primers:  harboring NheI restriction site (underlined); and  including BamHI restriction site (underlined). The PCR-amplified CPB-CTE gene was cloned into the NheI and BamHI sites of pAT153 vector downstream the CPA gene. Then CPA-CPB-CTE fusion gene was digested with HindIII and BamHI restriction sites and cloned downstream of the A2 gene in the pGEM7zf(+) vector. After sequence confirmation, the A2-CPA-CPB-CTE fusion gene was subcloned into the EcoRI and BamHI sites of vector pEGFP-N3 upstream of the GFP gene to generate pEGFP-A2-CPA-CPB-CTE, and the correct insert orientation was confirmed by restriction analysis. Then, the A2-CPA-CPB-CTE-GFP fragment was subcloned into the BglII and NotI sites of vector pLEXSY-NEO2  to generate pLEXSY-A2-CPA-CPB-CTE-GFP. Also the A2-CPA-CPB-CTE fragment was subcloned into the EcoRI and BamHI sites of vector pcDNA3.1(−)  to generate pcDNA-A2-CPA-CPB-CTE as a DNA vaccine. pcDNA-A2-CPA-CPB-CTE plasmid was purified by ion exchange chromatography with Endofree Mega kit .The L. tarentolae Tar II (ATCC 30267) strain was grown at pH 7.2 and 26°C in M199 medium (Sigma) supplemented with 5% heat-inactivated fetal calf serum , 40 mM HEPES, 0.1 mM adenosine, 5 µg/ml hemin and 50 µg/ml gentamicin. For transfection, 4×107 log-phase parasites were washed and re-suspended in 300 µl of electroporation buffer  and mixed with 50 µl H2O containing 15 µg of linearized pLEXSY-A2-CPA-CPB-CTE-GFP with SwaI, stored on ice for 10 min, and electroporated  at 450 V and 500 µF as described previously Leishmania promastigotes was evaluated by Epi-fluorescent microscopy for up to 3 months .The ssu locus was confirmed by diagnostic PCR using genomic DNA of transgenic strains as a template extracted by GF-1 Genomic DNA extraction kit . We performed diagnostic PCR  with ssu forward primer F3001 (5′-GATCTGGTTGATTCTGCCAGTAG-3′) and reverse primer A1715 hybridizing within the 5′UTR of the target gene (5′-TATTCGTTGTCAGATGGCGCAC-3′) according to the LEXSY Kit protocol . Primer pairs including one primer hybridizing within the expression cassette and one primer hybridizing to the ssu sequence not present in the plasmid were used. Integration of the expression cassette into the ssu locus yielded a 1 kb fragment that was not obtained in the control reactions with the genomic DNA of L. tarentolae wild type.Integration of the expression cassette into the L. tarentolae-GFP and wild type L. tarentolae genomic DNA were digested with the appropriate restriction enzymes (BglII/NotI). DNA was then resolved on 0.7% agarose before being separated and transferred onto membrane according to standard procedures GFP ORF was used by incorporating radiolabeled dCTP using Klenow enzyme. The reaction was finished by addition of 1 µl EDTA (0.5 M) and subsequently, the probe was purified by passing through a Sephadex resin. The membrane was then washed once with Wash buffer 1  at 25°C for 30 min, then 2 times at 65°C with Wash buffer 2  for 15 min. The membrane was then exposed for overnight and developed by a Konicka Minolta developer.For Southern blot analysis, 5 µg of transgenic -CTE and GFP cDNAs, PCR reactions were carried out using specific primer pairs to amplify each gene separately.RNA samples were extracted from promastigote forms using RNeasy kit (Qiagen) and treated with RNase-free DNase for 30 min at 37°C to eliminate any remaining DNA. cDNA synthesis was performed using the Qiagen Omniscript RT Kit from 1 µg of RNA. To detect the A2, CPA, CPBL. tarentolae-A2-CPA-CPB-CTE-GFP were examined for GFP expression by Epi-fluorescence microscopy. Promastigotes were centrifuged in 3000 rpm for 15 min and after washing once with PBS, cells were re-suspended in PBS and mounted on microscope slides. Expression of EGFP protein was evaluated by Epi-fluorescent microscopy . Also wild type (as negative control) and GFP expressing promastigote forms were analyzed for EGFP expression using flow cytometry. Parasites at two different growth phases (logarithmic and stationary phases) were centrifuged at 3000 rpm for 15 min, washed and then re-suspended at 106 cell/ml in PBS and stored on ice. Cells were analyzed on a FACS caliber flow cytometer  equipped with a 15 mV, 488 nm, air-cooled argon ion laser. 50,000 events were recorded and EGFP expression in transgenic L. tarentolae was measured in comparison with wild type (WT) parasites and L. tarentolae-GFP expressing parasites.Promastigote forms of L. tarentolae-A2-CPA-CPB-CTE-GFP were harvested by centrifugation at 3000 rpm for 15 min and washed in PBS. The pellets were immediately lysed in 2× SDS-PAGE sample buffer  on ice and then boiled for 5 min. Samples were then loaded on a 15% SDS-PAGE. The gels were transferred onto a nitrocellulose membrane using a Bio-Rad wet blotting system and incubated with blocking solution (PBS with 0.1% Tween 20 and 2.5% BSA) for 1 h. Washing was performed 3 times with 0.1% Tween 20 in PBS, and blots were incubated overnight with previously prepared rabbit anti-CPB polyclonal antibodies Promastigote forms of L. infantum strain MCAN/ES/98/LLM-877 was kindly provided by WHO collaborating center for leishmaniasis, Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain and kept virulent by continuous passage in hamsters. Amastigotes were isolated from the spleen of infected hamsters and cultured in NNN media in presence of 100 µg/ml of gentamicin. Stationary-phase promastigotes were harvested after 5–6 days by centrifugation , washed three times in PBS  and re-suspended at a concentration of 2×108 parasites/ml. This preparation was frozen and thawed (F/T) 10 times using liquid N2 and a 37°C water-bath and protein concentration was determined by bicinchoninic acid reagent . For infection, virulent promastigotes were harvested in the stationary phase, washed in PBS and injected (107) by the lateral tail vein into BALB/c mice.Six-week-old female BALB/c mice were obtained from the breeding stock maintained at the Pasteur Institute of Iran. The All mouse experiments including maintenance, animals' handling program and blood sample collection were approved by Institutional Animal Care and Research Advisory Committee of Pasteur Institute of Iran , based on the Specific National Ethical Guidelines for Biomedical Research issued by the Research and Technology Deputy of Ministry of Health and Medicinal Education (MOHME) of Iran (issued in 2005).-CTE-cSLN  vaccinated with 2×107 recombinant L. tarentolae-A2-CPA-CPB-CTE as prime and boost; group 3 (PBS as a control); group 4 [(empty vector pcDNA-cSLN (prime)/Live L. tarentolae wild type (boost) as a control)]; and group 5 (L. tarentolae Live/L. tarentolae Live) vaccinated with 2×107L. tarentolae wild type as prime and boost and used as a control. All groups were immunized via footpad. Booster immunization was carried out 3 weeks following the prime immunization. Three weeks after the last immunization, all animals were challenged with 107 stationary phase L. infantum promastigotes by lateral tail vein.Two independent immunization experiments were carried out in five groups of mice (n = 15 per treatment at each time point) and all tests were done in duplicate or triplicate (number of mice per group/time point n = 2–3). Results are shown as mean±S.E. of measures obtained from 4–6 mice in different groups. Group 1 (DNA cSLN/Live) immunized with pcDNA-A2-CPA-CPBLeishmania F/T at two different time points: before challenge and 5 weeks after challenge. Briefly, 96-well plates (Greiner) were coated with rA2, rCPA and rCPB or L. tarentolae-A2-CPA-CPB-CTE or L. infantum F/T, all at10 µg/ml, overnight at 4°C. Plates were blocked with 100 µl of 1% BSA in PBS at 37°C for 2 h to prevent nonspecific binding. Sera were added (with dilution of 1∶100) and incubated 2 h at 37°C. After three washes, Goat Anti-Mouse IgG1-HPR  or Goat Anti-Mouse IgG2a -HPR  were added and incubated for 2 h at 37°C. After four washes, plates were incubated for 30 min at 37°C with Peroxidase Substrate System  as substrate. Reactions were stopped with 1% SDS and the absorbance was measured at 405 nm.Serum samples were analyzed by ELISA for specific antibodies including IgG1 and IgG2a against either rA2, rCPs or 4Cl, 10 mM KHCO3 and 0.1 mM Na2EDTA), splenocytes were washed and re-suspended in complete RPMI medium . Cells were then seeded at a density of 3.5×106 cells/ml in the presence of rA2 (10 µg/ml), rCPA (10 µg/ml) and rCPB (10 µg/ml), or L. infantum F/T (25 µg/ml), or L. tarentolae-A2-CPA-CPB-CTE F/T (25 µg/ml), or medium alone. Concanavalin A  was also used in all experiments as the positive control. Plates were incubated for 24 h for IL-2 measurement and 5 days for IFN-γ and IL-10 measurement at 37°C in 5% CO2 humidified atmosphere. The IL-2, IFN-γ and IL-10 production in supernatants of splenocyte cultures was measured by sandwich ELISA kits , according to the manufacturer's instructions. The minimum detectable dose of mouse IFN-γ, IL-10 and IL-2 is typically less than 2, 4 and 7 pg/mL, respectively. All experiments were run in duplicates.Three mice from each group were sacrificed before challenge and also at 4 and 8 weeks after challenge and spleens were homogenized. After red blood cell lysis using ACK lysis buffer  ethylenediamine dihydrochloride, 1% sulfanil amide in 5% HDrosophila medium  supplemented with 20% heat-inactivated fetal calf serum and gentamicin (0.1%). Under sterile conditions, serial dilutions ranging from 1 to 10−20 were prepared in wells of 96 well microtitration plates. After 7 and 14 days of incubation at 26°C, plates were examined with an inverted microscope at a magnification of 40×. The presence or absence of mobile promastigotes was recorded in each well. The final titer was the last dilution for which the well contained at least one motile parasite. The number of parasites per gram was calculated in the following way: parasite burden = −log10 (parasite dilution/tissue weight) Two mice from each group were sacrificed at 2, 4, 8 and 12 weeks after challenge and parasite burden was determined as follows. A piece of spleen and liver was excised, weighed and then homogenized with a tissue grinder in 2 ml of Schneider's L. infantum named as RV1 and RV2  were used L. infantum genomic DNA was used in 10-fold dilutions corresponding to 2×105 parasites and used in real time PCR to draw the standard curve. For quantification of parasites in tissues, 300 ng of DNA was subjected to the reaction containing 5 pmol of each forward and reverse primers, 12.5 µl Qiagen QuantiFast SYBR Green Master Mix in total volume of 25 µl. All reactions were performed in duplicate. Conditions for PCR amplification were as follows: 95°C for 10 min; 40 cycles consisting of 95°C for 15 s, 58°C for 30 s, and 72°C for 40 s. Specific amplification of the target region was confirmed by gel electrophoresis of the PCR products.Furthermore, real time PCR was used to quantify parasite burden in spleen and liver 4 weeks after challenge for vaccinated groups (G1 and G2) and PBS control group (G3). Two mice from each group were sacrificed and genomic DNA was extracted from 10 mg of spleen and 30 mg of liver tissues using DNeasy Blood & Tissue kit (Qiagen). Two set of primers targeting a region of kinetoplastid minicircle DNA of Liver, spleen and bone marrow tissues of two animals from each group at 4, 8, and 14 weeks after challenge were fixed by 10% neutral-buffered formalin for 24 h, dehydrated by immersion in increasing concentrations of ethanol  and then xylene was added before embedding in paraffin wax. Four-micrometer-thick slides were prepared from paraffin blocks and were stained with hematoxylin and eosin (H&E) method. The slides were examined with an Olympus microscope (BX41) and photos were prepared by a DP11 digital camera (Olympus). The slides were reviewed by a pathologist who was not aware of the original treatment of the groups. The parenchyma of the liver was assessed for hepatocyte damage including fatty change, hydropic changes, cholestasis, liver cell necrosis and regenerative changes. The portals were assessed for degree of inflammation and interface hepatitis. Both areas were evaluated for presence of granuloma, neutrophils, plasma cells and lymphocytes as well as intracellular parasites. The micro-architecture of spleen composed of lymphoid follicles, splenic cord, and parafollicular area were evaluated. Presence of granuloma, giant cells and neutrophils were also assessed.Statistics were performed using Graph-Pad Prism 5.0 for Windows  as well as SPSS version 18. All the data were analyzed with one way ANOVA (Multiple-comparison Tukey post Hoc test) and when required with a Student's t-test. The correlation between the ratio of IFN-γ/IL-10 production and differences in parasite burden at weeks 4 and 8 were calculated using Spearman Correlation method (2 tailed). The area under the curve (AUC) of parasite burden graphs was calculated using Graph-Pad Prism 5.0 software program. The results were considered statistically significant when p<0.05.L. tarentolae stably expressing the L. donovani A2 antigen along with L. infantum cysteine proteinases CPA and CPB without its unusual C-terminal extension (CPB-CTE) as a tri-fusion gene (A2-CPA-CPB-CTE) together with the EGFP gene were generated by introducing the linearized pLEXSY-A2-CPA-CPB-CTE-EGFP vector into the 18S rRNA ssu locus of L. tarentolae (ssu locus was confirmed both by genomic PCR (-CTE-EGFP by L. tarentolae at the mRNA level (L. tarentolae-A2-CPA-CPB-CTE-EGFP and L. tarentolae-EGFP parasites was confirmed by fluorescence microscopy (L. tarentolae-A2-CPA-CPB-CTE-EGFP-expressing parasites was decreased in logarithmic phase (72%) as compared to L. tarentolae-EGFP (93%) with decreasing in mean fluorescent intensities . It was shown previously that this formulation facilitates the uptake by dendritic cells and macrophages, thereby enhancing antigen expression, processing and presentation and resulting in stronger immune effects -CTE/Live L. tar A2-CPA-CPB-CTE (G1) and L. tar A2-CPA-CPB-CTE/L. tar A2-CPA-CPB-CTE (G2) prime-boosting regiments drastically (p<0.01) reduced the infection levels in both the liver . The liver parasite load  due most likely to high inoculum (107). In the spleen, the highest parasite burden in control groups  was observed at 12 weeks after challenge and the organ remained chronically infected . Thus, the G2 vaccination regimen protects against infection better than G5 (Live L. tar/Live L. tar). Also, the spleen AUC of DNA/Live (G1) and Live/Live (G2) parasites indicate significant differences with the controls G4 and G5  only 8 weeks after challenge in the spleen of all five groups following stimulation with rA2-rCPA-rCPB, F/T L.tarA2-CPA-CPB-CTE-EGFP and F/T L. infantum antigens (n = 6 at each mentioned period L. tar A2-CPA-CPB-CTE-EGFP lysate and L. infantum lysate) elicited a significantly higher IFN-γ production than control groups G3, G4 and G5. The production of IFN-γ in response to rA2-rCPA-rCPB antigens reached the highest level (524±16.4 pg/ml) at 4 weeks after challenge in G2 (Live/Live). This was significantly (p<0.05) different from G1 (DNA/Live) (404±19.9 pg/ml). IFN-γ levels were maintained high even 8 weeks post-challenge in both G1 (404.5±101 pg/ml) and G2 (331±27 pg/ml)  before challenge but this was not significantly different from G2 ) and spleen cells (−.640*(p = .046)) stimulated with F/T L. infantum at 4th week after challenge. This means that higher amount of IFN-γ/IL-10 ratio resulted in lower parasite burden in both spleen and liver at 4th week after challenge. The IFN-γ/IL-10 ratio was drawn against the parasite burden in both spleen and liver at 4th week after challenge with individual values of each group (five groups) in liver and spleen as well as their mean, and the negative slops in both graphs are in concordance with this fact (The above experiments demonstrate that immunization with recombinant antigens . In addi7 pg/ml) . Stimula from G2 . IFN-γ l from G2 . The pro from G2 . We furt from G2 . Indeed,his fact .IL-2 production that is important for lymphocyte proliferation was higher in G1 and G2 than in control groups . SignifiL. tarA2-CPA-CPB-CTE-GFP and F/T L. infantum antigens at 8 weeks after challenge  upon stimulation with rA2-rCPA-rCPB, F/T hallenge .L. infantum specific IgG1 and IgG2a were higher in vaccinated groups G1 and G2 compared to the control groups. Although G1 shows higher amount of rA2-rCPA-rCPB specific IgG2a, there were no significant differences with control groups. Also, increased amount of F/T L. infantum specific IgG1 was seen in G5 at 5 weeks after challenge. Interestingly, in G2 vaccinated mice with Live L.tar A2-CPA-CPB-CTE-GFP/Live L.tar A2-CPA-CPB-CTE-GFP, a higher amount of F/T L. tar A2-CPA-CPB-CTE-GFP specific IgG1 and IgG2a was detected in comparison to G1 and G4 vaccinated with DNA/Live modality  (th week and not seen later. We could not find parasites in the spleen in vaccinated groups (G1 and G2) but they could be seen in nearly all non-vaccinated groups  after challenge to collect liver, spleen and bone marrow samples for histopathological analysis. In spleen, splenic cords were normal and somewhat thickened in vaccinated groups but were thin and in some cases were disappeared in other groups. Normal follicle formation of the spleen was noted in G1 and G2 in 4 and G5) . Absence and G5) . Spleen th. Plasma cells were only seen in these groups. Interface hepatitis was not a significant finding. Parasites in the portals were only seen in non-vaccinated groups (G3 and G5) in 8th week (data not shown).In the liver, mononuclear cell infiltration in portals was more prominent in non-vaccinated groups at week 14th week, increased significantly in 8th week and decreased at week 14. The severity of this inflammatory response at 4th week was higher in G3 compared to the other groups (15–16/10 hpf (high power field) v.s 2–4/10 hpf of vaccinated groups, p<0.05) . There was no significant difference in neutrophil infiltration, fatty changes, giant cell formation, and kupffer cell hyperplasia between groups (data not shown). Liver cell necrosis was minimal and no significant regenerative changes were noted (data not shown). No parasites were found in bone marrow macrophages and no significant changes in bone marrow of all examined mice were observed (data not shown).In all groups, lobular inflammation was seen at 4 p<0.05) . Inflammhallenge . Portal 4th week . ParasitLeishmania vaccine development has been proven to be a difficult and challenging task and is hampered by an inadequate knowledge of disease pathogenesis, the complexity of immune responses needed for protection, and the cost of vaccine development L. major murine model, this dichotomy seems inadequate in human and murine forms of VL. Type 1 immune responses are suppressed by IL-10 and TGF-β +CD25+ T cells were found responsible for TGF-β and CD4+CD25−Foxp3− T cells for IL-10 production Leishmania vaccine researchers that parasite persistence and exposure to complex antigens in the right context over time may be important for effective protective response and could be achieved by live attenuated parasites immunization. However, reversion of these parasites to the virulent form restricts their use in vitro culture, γ-irradiation, selection for temperature sensitivity, or chemical mutagenesis) did not elicit a protective immunity Leishmania genome through discovery of new genes involved in parasite growth and survival revives the potential of a live attenuated parasite vaccine with reduced risk of reversion. In this context, several successful attenuated lines of L. donovani, such as a partial knockout of A2-A2rel gene clusters with attenuated virulence in mice /−−) knockout with reduced parasite survival in macrophages Leishmania vaccines, which most closely mimic the natural course of infection, could be very beneficial. On the other hand, return back to virulence may occur, and there is an increasing need to develop new safer live vaccine vectors that are capable of enhancing antigen presentation and eliciting potent immune responses without the risk of disease development in humans. Consequently, a live nonpathogenic to humans parasitic vector consisting of a lizard parasitic protozoan, L. tarentolae, has been introduced by Breton et al. as a candidate vaccine against visceral leishmaniasis L. tarentolae strain expressing the L. donovani A2 gene elicited a strong protective immunity against virulent L. infantum challenge L. infantum cysteine proteinases type I and II as part of a prime-boost strategy induced a strong parasite-specific Th1 response and conferred protection against parasite challenge Although preventive vaccines are recognized as the best and most cost-effective protection measure against pathogens, no effective vaccines are available to control leishmaniasis. L. tarentolae strain stably expressing three candidate antigens as part of a tri-gene fusion, including the L. donovani A2 and L. infantum CPA and CPB genes and to evaluate its potential as a candidate live vaccine against VL using different prime-boost vaccine modalities. Immunization of BALB/c mice with recombinant L. tarentolae A2-CPA-CPB-CTE as a DNA/Live or a Live/Live prime-boost regimen (groups G1 and G2) elicited a significant protective immunity against a high dose virulent L. infantum challenge. Moreover, mice immunized with these modalities did control the parasite propagation both in the liver and the spleen at 4 weeks but also 12 weeks after challenge. It is worth mentioning that tissue parasite loads were measured with the limiting dilution assay that is semi-quantitative and may give inconsistent results due to medium and incubation conditions. Hence, for more accurate measurements of parasite burden considering both LDA and qPCR is highly recommended. Therefore at week 4 after infectious challenge, we also confirmed the quantity differences between vaccinated groups (G1 and G2) and PBS control group (G3) by qPCR in both liver and spleen. Interestingly, the obtained results for this specific time point are in accordance with semi-quantitative LDA.As a follow-up of our previous work, the objective of this study was to generate a recombinant L. tarentolae A2-CPA-CPB-CTE revealed the induction and secretion of IFN-γ and IL-10, although IFN-γ was much higher than IL-10. Determining the main source of IL-10 production has some beneficial results for understanding mechanisms of resistance and susceptibility to VL. Here, in immunized G1 and G2 groups, we detected high IL-10 production, especially at 8 weeks after challenge, upon stimulation with L. infantum lysate as a recall antigen. The sources of IL-10 in human VL have not been defined yet. IL-10 can be produced by several cell types such as monocytes, macrophages, B cells and CD4+ T cells. It has been described that several subpopulations of IL-10 producing CD4+ T cells have the ability to inhibit the response of other T cells. One of the important sources of IL-10 in the suppression of anti-leishmanial immunity in human VL is T regulatory (T reg) cells that arise from CD25−Foxp3− T cells + CD25− depleted cells that produce both IFN-γ and IL-10 cytokines. Simultaneous production of IFN-γ and IL-10 by human T cell clones can be induced by IL-12 In our experimental system, immunization of BALB/c mice with recombinant L. tarentolae A2-CPA-CPB-CTE was ∼5.1- to 11.2-fold higher at 4 and 8 weeks post-challenge than mice immunized with wild type L. tarentolae only. The same result was obtained with splenocytes stimulated with other F/T antigens. Also, histopathological studies confirmed the Spearman correlation between the ratio of IFN-γ/IL-10 production and differences in parasite burden in liver and spleen cells as no parasites were seen in the liver parenchyma and spleen at 4th week post-challenge in the vaccinated groups G1 and G2, whereas they were easily detected in the non-vaccinated control groups. In addition, the level of both IgG1 and IgG2a at different time points before and after challenge was not significantly different between the vaccinated groups. Thus, cytokine data along with antibody titration indicated that vaccination with DNA/Live L. tarentolae A2-CPA-CPB-CTE and Live/Live L. tarentolae A2-CPA-CPB-CTE induce a clearly stronger Th1 response.The IFN-γ/IL-10 ratio in splenocytes stimulated with rA2-rCPA-rCPB from mice immunized with the recombinant L. tarentolae A2-CPA-CPB-CTE, especially when administered as a Live/Live modality, induces a strong humoral and cellular immune response in addition to NO generation in response to rA2-rCPA-rCPB, F/T L. tarentolae A2-CPA-CPB-CTE and F/T L. infantum specific stimulators at 8 weeks after infectious challenge with L. infantum. Possibly, the cumulative effect of IFN-γ along with iNOs-mediated parasite killing is responsible for this response. Furthermore, it has been reported that one of the major factors contributing to healing of leishmaniasis is the development of strong CMI (cell-mediated immunity) response like IFN-γ and NO production L. tarentolae A2-CPA-CPB-CTE and F/T L. infantum also supports the up-regulation of inducible NO synthase by TH1 cells and confirms that the NO-mediated macrophage effector mechanism is critical in the control of parasite replication in these animals. Our results showed that the nitrite (NO2) levels are in direct correlation with IFN-γ production in these groups and that higher production of IFN-γ was obtained after 56 days of infection, indicating a possible control of the parasites' persistence and suggesting that the control of L. infantum infection may occur by a NO-dependent pathway.Although it has been suggested that the control of splenic parasite burden is likely to be mediated through a NO-dependent or a NO-independent pathway 7L. infantum promastigotes, control groups exhibited severe histopathological alterations in both the spleen and liver at the peak of parasite burden. Once in the liver, the development of cell-mediated immune responses is essential for the clearance of L. infantum parasites. In contrast, the spleen ultimately becomes the site of parasite persistence L. infantum infection than the liver L. infantum, which results in liver parasite clearance. However, disruption of the splenic architecture accompanied by lymphoid depletion only in control groups results in spleen parasite persistence.It has been described previously that during the early stages of visceral infection in BALB/c mice, parasites multiply in large numbers in the liver; however, once the infection becomes chronic, hepatic parasite loads tend to decrease, while parasitism in the spleen and BM tends to increase L. tarentolae A2-CPA-CPB-CTE candidate vaccine protects mice against visceral leishmaniasis when administered as a prime-boost modality more than L. tarentolaeL. tarentolae A2 L. infantum in dogs as an important outbreed animal model for VL research.Overall, our results on cytokine production, humoral responses, parasite burden and histopathological studies support that immunization with the novel recombinant Figure S1Generation of a recombinant L. tarentolae-CTE-EGFP tri-fusion gene. strain expressing the A2-CPA-CPB (A) Schematic representation of the linearized pLEXSY-A2-CPA-CPB-CTE-EGFP construct containing two regions of homology to the rRNA locus of L. tarentolae 5′ssu and 3′ssu for genomic integration by homologous recombination following transfection into L. tarentolae. (B) PCR analysis to confirm integration of the A2-CPA-CPB-CTE-EGFP cassette into the ssu locus (lane 1). Wild type L. tarentolae was used as a negative control (lane 2). (C) Southern blot hybridization of DNA extracted from L. tarentolae promastigotes and digested with BglII/NotI with a GFP-labeled probe. Lanes 1, L. tarentolae wild type; 2, a 2.8-kb region of A2-CPA-CPB-CTE-EGFP; and 3, a<1-kb region of EGFP for L. tarentolae-EGFP as a positive control. (D) RT-PCR analysis of A2, CPA, -CTECPB and EGFP genes from cDNA of L. tarentolae-A2-CPA-CPB-CTE-EGFP. The 708 bp band represents the A2 PCR product on cDNA template (lane 1), a 687 bp band represents the CPA PCR product (lane 3), a 639 bp band represents the CPB-CTE PCR product (lane 5), and a 735 bp band represents the EGFP PCR product (lane 7). No bands were detected for the PCR reaction on RNA templates for A2, CPA, -CTECPB and EGFP genes, respectively in lanes 2, 4, 6 and 8.(TIF)Click here for additional data file.Figure S2Quantification of parasites in liver and spleen at 4 weeks after challenge by real time PCR assays. The parasite number in the liver (A) or spleen (B) was evaluated by Real time PCR at 4 weeks post- infection for vaccinated groups (G1 and G2) and PBS control group (G3). G1 [vaccinated with DNA A2-CPA-CPB-CTE-cSLN (prime) and Live L. tarentolae-A2-CPA-CPB-CTE (boost)]; G2 [vaccinated with Live L. tarentolae-A2-CPA-CPB-CTE (prime) and Live L. tarentolae-A2-CPA-CPB-CTE (boost)] and G3 (control PBS) groups. The number of independent repeats was two and all tests were done in duplicate (number of mice per group/time point n = 2). The result is shown here as mean±S.E. of measures obtained from 4 mice of each group. The asterisk indicates the significant difference between values at the indicated time points as determined by Student's test .(TIF)Click here for additional data file.Figure S3th week after challenge.IFN-γ/IL-10 ratio against parasite burden in liver and spleen at 4 IFN-γ/IL-10 ratio inversely correlates with the parasite burden at 4 weeks after challenge with individual values of each group (five groups) in liver (A) and spleen (B) (each data series represented by different colors). Inset graphs in both A and B shows their mean with significant p value (p<0.05) and r2. Groups G1 [vaccinated with DNA A2-CPA-CPB-CTE-cSLN (prime) and Live L. tarentolae-A2-CPA-CPB-CTE (boost)]; G2 [vaccinated with Live L. tarentolae-A2-CPA-CPB-CTE (prime) and Live L. tarentolae-A2-CPA-CPB-CTE (boost)]; G3 (control PBS); G4 [DNA vector alone (prime) and Live L. tarentolae wild type (boost)], and G5 [Live L. tarentolae wild type (prime) and Live L. tarentolae wild type (boost)].(TIF)Click here for additional data file.Figure S4th week after challenge.Inflammatory cell infiltration in the liver parenchyma of all groups at 4 The liver parenchyma were stained with hematoxylin and eosin (H&E). G1 to G5 groups are as indicated in (TIF)Click here for additional data file.


The pandemic 2009-H1N1 influenza virus circulated in the human population and caused thousands deaths worldwide. Studies on pandemic influenza vaccines have shown that T cell recognition to conserved epitopes and cross-reactive T cell responses are important when new strains emerge, especially in the absence of antibody cross-reactivity. In this work, using HLA-B*4405 and DM1-TCR structure model, we systematically generated high confidence conserved 2009-H1N1 T cell epitope candidates and investigated their potential cross-reactivity against H5N1 avian flu virus.Molecular docking analysis of differential DM1-TCR recognition of the 2009-H1N1 epitope candidates yielded a mosaic epitope (KEKMNTEFW) and potential H5N1 HA cross-reactive epitopes that could be applied as multivalent peptide towards influenza A vaccine development. Structural models of TCR cross-recognition between 2009-H1N1 and 2004-H5N1 revealed steric and topological effects of TCR contact residue mutations on TCR binding affinity.The results are novel with regard to HA epitopes and useful for developing possible vaccination strategies against the rapidly changing influenza viruses. Yet, the challenge of identifying epitope candidates that result in heterologous T cell immunity under natural influenza infection conditions can only be overcome if more structural data on the TCR repertoire become available. In 2009, the outbreak of a new swine-origin strain of influenza A H1N1 caused widespread human infection  and followed by docking of the HLA/epitope complex as a ligand to DM1-TCR [PDB:3DXA]. The binding ability of the predicted epitopes was further analyzed using ClusPro v.2 [The pipeline included docking the epitopes to HLA-B*4405 [PDB:sPro v.2 ,26.ClusPro v.2 is a web-based automated docking program performing a multistage protocol: rigid PIPER docking, filtering and clustering of docked conformations, and stabilizing using Monte Carlo simulations . During ff99SB [2 on the whole complex during the first 1,000 steps under restrained conditions, while initially minimizing positions of solvent and sodium ions. For the subsequent 2,500 steps of minimization, we removed this restraint. The constant volume was set during both the 2 stages of the minimization process. In the heating stage of 20 ps, we restrained the complex again, but with only 10 kcal/molÅ2 to avoid wild fluctuations in the structure. We allowed the system to heat up from 0 K to 300 K and applied the Langevin temperature equilibration scheme to control the temperature. Then, a short equilibration stage (1ns) without the restraints was performed in constant pressure of 1 atm and at 300 K. We used SHAKE in both heating and equilibration stages to constrain bonds that involves hydrogen. As a result, complexes obtained from the MD simulation above were considered final bound conformations of the docked TCR-HLA/epitope complexes in our study.Then, we performed molecular dynamics (MD) simulations using AMBER 10 force field ff99SB  to improSome abbreviations were used in the text: HA: (Hemagglutinin); HLA: (Human leukocyte antigen); TCR: (T cell Receptor); CTL: (Cytotoxic T Lymphocyte); MHC: (Major histocompatibility complex); TAP: (Transporter associated with antigen processing); DARS: (Decoys as the Reference States); MD: (Molecular Dynamics).The authors declare that they have no competing interests.CTTS carried out molecular docking experiments. CTTS, CS and CKK wrote the manuscript. All authors read and approved the final manuscript.This work was supported by Singapore MOE AcRF Grant No: MOE2008-T2-1-1074 and Jardine OneSolution JOS-M4060054.Funding for publication of this work was supported by Singapore MOE AcRF Grant No: MOE2008-T2-1-1074 and Jardine OneSolution JOS-M4060054.BMC Bioinformatics Volume 14 Supplement 2, 2013: Selected articles from the Eleventh Asia Pacific Bioinformatics Conference (APBC 2013): Bioinformatics. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcbioinformatics/supplements/14/S2.This article has been published as part of MAFFT multiple sequence alignment of pandemic strains (2009 - the top 7 sequences) HA proteins to HA (1918 - South Carolina) and WHO vaccine HA (the last 3 sequences).Click here for filePredicted HA T cell epitopes of H1N1 2009 strains compared to 1918 and WHO-1999-2006-2007 strains.Click here for filePhysicochemical properties of NetCTL-predicted HLA-B44 restricted T cell epitope candidates (A) and non-epitopes (B).Click here for fileResidues of domain α1, α2 of HLA-B*4405 interacting with DM1-TCR.Click here for file