The expression, processing and localization of polymorphic membrane proteins in Chlamydia pneumoniae strain CWL029

BACKGROUND: Chlamydiae are obligate intracellular bacteria, which are important human pathogens. Genome sequences of C. trachomatis and C. pneumoniae have revealed the presence of a Chlamydia specific gene family encoding polymorphic outer membrane proteins, Pmps. In C. pneumoniae the family comprises twenty-one members, which are all transcribed. In the present study, the expression, processing and localisation of the sixteen full-length Pmps in C. pneumoniae strain CWL029 have been further investigated by two-dimensional gel electrophoresis and immunofluorescence microscopy. RESULTS: Ten Pmps were identified in elementary bodies (EBs). Eight of these were investigated with respect to time dependent expression and all were found to be up-regulated between 36 and 48 hours post infection. Antibodies against Pmp6, 8, 10, 11 and 21 reacted with chlamydiae when infected cells were formalin fixed. Pmp6, Pmp20 and Pmp21 were found in cleaved forms, and the cleavage sites of Pmp6 and Pmp21 were identified. CONCLUSIONS: The Pmps are heavily up-regulated at the time of conversion of RB to EB, and at least ten Pmps are present in EBs. Due to their reaction in formalin fixation it is likely that Pmp6, 8, 10, 11 and 21 are surface exposed. The identified cleavage sites of Pmp6 and Pmp21 are in agreement with the theory that the Pmps are autotransporters

Distinct patterns of blood-stage parasite antigens detected by plasma IgG subclasses from individuals with different level of exposure to Plasmodium falciparum infections

International audienceBACKGROUND: In endemic regions naturally acquired immunity against Plasmodium falciparum develops as a function of age and exposure to parasite infections and is known to be mediated by IgG. The targets of protective antibodies remain to be fully defined. Several immunoepidemiological studies have indicated an association of cytophilic anti-parasite IgG with protection against malaria. It has been hypothesized that the initial antibody responses against parasite antigens upon first few Plasmodium falciparum infections is dominated by non-protective IgG2/IgG4 and IgM antibodies, which then gradually develop into protective response dominated by cytophilic IgG1 and IgG3 antibodies. METHODS: Naturally occurring IgG antibodies against P. falciparum blood-stage antigens were analysed from plasma samples collected from four groups of individuals differing in age and level of exposure to P. falciparum infections. Western Blot profiling of blood-stage parasite antigens displaying reactivity with individual plasma samples in terms of their subclass specificities was conducted. Parasite antigens detected by IgG were grouped based on their apparent molecular sizes resolved by SDS-PAGE as high molecular weight (≥ 70 kDa) or low molecular weight (< 70 kDa). The number of discernable low molecular weight parasite antigens detected by different IgG subclass antibodies from each plasma sample was recorded. Using Wilcoxons rank sum test these reactivities were compared amongst groups of individuals with different levels of exposure to P. falciparum infections. RESULTS: IgG4 and IgM antibodies in plasma samples from all groups detected very few parasite antigens. IgG2 antibodies from all groups detected a common pattern of high molecular weight parasite antigens. Cytophilic IgG subclasses in plasma samples from individuals with higher levels of exposure to P. falciparum infections distinctly detected higher numbers of low molecular weight parasite antigens. CONCLUSIONS: In the present study, there was no evidence for switching of antibody responses from non-cytophilic to cytophilic subclasses against blood-stage parasite antigens as a likely mechanism for induction of protective immunity against malaria

Cationic Liposomes Formulated with Synthetic Mycobacterial Cordfactor (CAF01): A Versatile Adjuvant for Vaccines with Different Immunological Requirements

It is now emerging that for vaccines against a range of diseases including influenza, malaria and HIV, the induction of a humoral response is insufficient and a substantial complementary cell-mediated immune response is necessary for adequate protection. Furthermore, for some diseases such as tuberculosis, a cellular response seems to be the sole effector mechanism required for protection. The development of new adjuvants capable of inducing highly complex immune responses with strong antigen-specific T-cell responses in addition to antibodies is therefore urgently needed. (cell-mediated/humoral) and malaria (humoral) immunization with CAF01-based vaccines elicited significant protective immunity against challenge.CAF01 is potentially a suitable adjuvant for a wide range of diseases including targets requiring both CMI and humoral immune responses for protection

Desarrollo sustentable en América Latina : oportunidades para la próxima década

Amyloid fibrils formed by the 29-residue peptide hormone glucagon at different concentrations have strikingly different morphologies when observed by transmission electron microscopy. Fibrils formed at low concentration (0.25 mg/mL) consist of two or more protofilaments with a regular twist, while fibrils at high concentration (8 mg/mL) consist of two straight protofilaments. Here, we explore the structural differences underlying glucagon polymorphism using proteolytic degradation, linear and circular dichroism, Fourier transform infrared spectroscopy (FTIR), and X-ray fiber diffraction. Morphological differences are perpetuated at all structural levels, indicating that the two fibril classes differ in terms of protofilament backbone regions, secondary structure, chromophore alignment along the fibril axis, and fibril superstructure. Straight fibrils show a conventional beta-sheet-rich far-UV circular dichroism spectrum whereas that of twisted fibrils is dominated by contributions from beta-turns. Fourier transform infrared spectroscopy confirms this and also indicates a more dense backbone with weaker hydrogen bonding for the twisted morphology. According to linear dichroism, the secondary structural elements and the aromatic side chains in the straight fibrils are more highly ordered with respect to the alignment axis than the twisted fibrils. A series of highly periodical reflections in the diffractogram of the straight fibrils can be fitted to the diffraction pattern expected from a cylinder. Thus, the highly integrated structural organization in the straight fibril leads to a compact and highly uniform fibril with a well-defined edge. Prolonged proteolytic digestion confirmed that the straight fibrils are very compact and stable, while parts of the twisted fibril backbone are much more readily degraded. Differences in the digest patterns of the two morphologies correlate with predictions from two algorithms, suggesting that the polymorphism is inherent in the glucagon sequence. Glucagon provides a striking illustration of how the same short sequence can be folded into two remarkably different fibrillar structures. (C) 2010 Elsevier Ltd. All rights reserved

CAF01-induced responses in TLR2, 3, 4, 7 and MyD88-deficient mice.

<p>A) MyD88-/- and B) TLR2, 3, 4, 7-/- mice as well as WT controls mice (n = 3) were vaccinated twice with 2 µg of Ag85B-ESAT-6 in CAF01 by footpad immunization. Two weeks after the last immunization, the inguinal lymphnodes were harvested and restimulated in vitro with 10 µg of Ag85B-ESAT-6. The release of IFN-γ was determined by ELISA.</p

Immune responses and protection induced by CAF01 in a TB model.

<p>C57BL/6 mice were immunized three times with 2 µg of Ag85B-ESAT-6 in CAF01 or Al(OH)₃. A) Three weeks after the last immunization, mice were bled by periorbital puncture and individual sera tested for Ag85B-ESAT-6 IgG1, B) IgG2b or C) IgG2c by ELISA (n = 6). D) Individual cultures of splenocytes (n = 3) were harvested at the same time point and re-stimulated in vitro with different concentrations of the Ag85B-ESAT-6. The release of IFN-γ was determined by ELISA. Six weeks after the last immunization, mice were challenged by the aerosol route with virulent <i>M. tuberculosis</i>. Six weeks postchallenge, mice were sacrificed and the bacterial burden (CFU) measured in the E) lungs or F) spleen (expressed as log₁₀ CFU). As a positive control group, a group of mice received a BCG vaccination ten weeks before challenge. Data shown are mean values of six mice ± SEM. G) At different time points after infection, mice were sacrificed and the CFU measured in the lungs. Values marked with an asterisk are significantly different (*, <i>p</i><0.05; **, <i>p</i><0.01, <i>p</i><0.001) compared to naïve controls as assessed by ANOVA and Dunnett's multiple comparison test.</p

Comparison of CAF01 with other adjuvants.

<p>C57BL/6 mice (n = 4) were immunized three times with 100 µg of OVA in CAF01, Al(OH)3, Montanide, MPL, CFA/2×IFA boosting with a two-week interval. A) Three weeks after the last immunization, the number of OVA-specific cells was assessed by IFN-γ ELISPOT. Mean spot-forming units (SFU) upon stimulation with 5 µg of OVA per million cells ± SEM for each group is shown. B) SFU per million cells ± SEM upon stimulation with 5 µg of OVA CD4 T cell epitope. C) SFU per million cells ± SEM upon stimulation with 5 µg of OVA CD4 T cell epitope. D) Sera were analysed for the presence of OVA-specific IgG1, E) IgG2b, F) IgG2c antibodies by ELISA. Mean EC50 ± SEM is shown. Values marked with an asterisk are significantly different (*, <i>p</i><0.05; **, <i>p</i><0.01, <i>p</i><0.001) compared to naïve controls as assessed by ANOVA and Dunnett's multiple comparison test.</p

Immune responses and protection induced by CAF01 in a malaria blood-stage model.

<p>BALB/c mice were immunized three times with 10 µg of MSP1 in CAF01 or Al(OH)₃. One week after the last immunization, mice were bled by periorbital puncture and individual sera tested for MSP1-specific A) IgG1 or B) IgG2a by ELISA (n = 5). C) Individual cultures of splenocytes (n = 3) were harvested at the same time point and re-stimulated in vitro with different concentrations of MSP1. The release of IFN-γ was determined by ELISA. D and E) Three weeks after the last immunization, mice were challenged by the i.p. route with <i>Plasmodium yoelli</i> and the number of infected red blood cells measured at various time points during infection. Data shown are mean values of five mice ± SEM. Values marked with an asterisk are significantly different (*, <i>p</i><0.05; **, <i>p</i><0.01, <i>p</i><0.001) compared to Al(OH)₃-vaccinated as assessed by t-test.</p