Metatranscriptomic assessment of burn wound infection clearance

Peer reviewe

Polyantigenic Interferon-γ Responses Are Associated with Protection from TB among HIV-Infected Adults with Childhood BCG Immunization

Surrogate immunologic markers for natural and vaccine-mediated protection against tuberculosis (TB) have not been identified. HIV-infected adults with childhood BCG immunization entering the placebo arm of the DarDar TB vaccine trial in Dar es Salaam, Tanzania, were assessed for interferon gamma (IFN-γ) responses to three mycobacterial antigen preparations--secreted Mycobacterium tuberculosis antigens 85 (Ag85), early secretory antigenic target 6 (ESAT-6) and polyantigenic whole cell lysate (WCL). We investigated the association between the number of detectable IFN-γ responses at baseline and the subsequent risk of HIV-associated TB. During a median follow-up of 3.3 years, 92 (9.4%) of 979 placebo recipients developed TB. The incidence of TB was 14% in subjects with no detectable baseline IFN-γ responses vs. 8% in subjects with response to polyantigenic WCL (P = 0.028). Concomitant responses to secreted antigens were associated with further reduction in the incidence of HIV-associated TB. Overall the percentage of subjects with 0, 1, 2 and 3 baseline IFN-γ responses to mycobacterial preparations who developed HIV-associated TB was 14%, 8%, 7% and 4%, respectively (P = 0.004). In a multivariate Cox regression model, the hazard of developing HIV-associated TB was 46% lower with each increment in the number of detectable baseline IFN-γ responses (P<0.001). Among HIV-infected adults who received BCG in childhood and live in a TB-endemic country, polyantigenic IFN-γ responses are associated with decreased risk of subsequent HIV-associated TB. ClinicalTrials.gov NCT0052195

Cell-mediated immune responses in a Chlamydia pneumoniae infection model

Väitöskirj

Production of Chlamydia pneumoniae Proteins in Bacillus subtilis and Their Use in Characterizing Immune Responses in the Experimental Infection Model

Due to intracellular growth requirements, large-scale cultures of chlamydiae and purification of its proteins are difficult and laborious. To overcome these problems we produced chlamydial proteins in a heterologous host, Bacillus subtilis, a gram-positive nonpathogenic bacterium. The genes of Chlamydia pneumoniae major outer membrane protein (MOMP), the cysteine-rich outer membrane protein (Omp2), and the heat shock protein (Hsp60) were amplified by PCR, and the PCR products were cloned into expression vectors containing a promoter, a ribosome binding site, and a truncated signal sequence of the α-amylase gene from Bacillus amyloliquefaciens. C. pneumoniae genes were readily expressed in B. subtilis under the control of the α-amylase promoter. The recombinant proteins MOMP and Hsp60 were purified from the bacterial lysate with the aid of the carboxy-terminal histidine hexamer tag by affinity chromatography. The Omp2 was separated as an insoluble fraction after 8 M urea treatment. The purified proteins were successfully used as immunogens and as antigens in serological assays and in a lymphoproliferation test. The Omp2 and Hsp60 antigens were readily recognized by the antibodies appearing after pulmonary infection following intranasal inoculation of C. pneumoniae in mice. Also, splenocytes collected from mice immunized with MOMP or Hsp60 proteins proliferated in response to in vitro stimulation with the corresponding proteins

Identification of Chlamydia pneumoniae-Derived Mouse CD8 Epitopes

Chlamydia pneumoniae is a common intracellular human pathogen that has been associated with several severe pathological conditions, including coronary heart disease and atherosclerosis. There is no vaccine against C. pneumoniae infection, but CD8(+) T cells have been shown to be crucial for protection during experimental infection. However, the effector functions and epitope specificity of the protective CD8(+) T cell remain unknown. The aim of this study was to identify C. pneumoniae-derived mouse CD8 epitopes by using a recent epitope prediction method. Of four C. pneumoniae proteins (the major outer membrane protein, outer membrane protein 2, polymorphic outer membrane protein 5, and heat shock protein 60), 53 potential CD8(+) T-cell epitopes were predicted by H-2 class I binding algorithms. Nineteen of the 53 peptides were identified as CD8 epitopes by testing for induction of a cytotoxic response after immunization. To test whether the predicted epitopes are naturally processed and presented by C. pneumoniae-infected cells, we generated a panel of seven peptide-specific cytotoxic T lymphocyte lines that were subsequently tested for recognition of C. pneumoniae-infected target cells. By using this strategy, we were able to identify three C. pneumoniae CD8 epitopes that were, indeed, processed and presented on infected cells. Identification of these natural CD8 epitopes provides tools for characterization of CD8(+) T-cell function in vivo and generation of epitope-specific prevention strategies