Elizabeth R. Jarman

BCG-induced increase in interferon-gamma response to mycobacterial antigens and efficacy of BCG vaccination in Malawi and the UK: two randomised controlled studies

BACKGROUND The efficacy of BCG vaccines against pulmonary tuberculosis varies between populations, showing no protection in Malawi but 50-80% protection in the UK. To investigate the mechanism underlying these differences, randomised controlled studies were set up to measure vaccine-induced immune responsiveness to mycobacterial antigens in both populations. METHODS 483 adolescents and young adults in Malawi and 180 adolescents in the UK were tested for interferon-gamma (IFN-gamma) response to M tuberculosis purified protein derivative (PPD) in a whole blood assay, and for delayed type hypersensitivity (DTH) skin test response to tuberculin PPD, before and 1 year after receiving BCG (Glaxo 1077) vaccination or placebo or no vaccine. FINDINGS The percentages of the randomised individuals who showed IFN-gamma and DTH responses were higher in Malawi than in the UK pre-vaccination-ie, 61% (331/546) versus 22% (47/213) for IFN-gamma and 46% (236/517) versus 13% (27/211) for DTH. IFN-gamma responses increased more in the UK than in Malawi, with 83% (101/122) and 78% (251/321) respectively of the vaccinated groups responding, with similar distributions in the two populations 1 year post-vaccination. The DTH response increased following vaccination in both locations, but to a greater extent in the UK than Malawi. The IFN-gamma and DTH responses were strongly associated, except among vaccinees in Malawi. INTERPRETATION The magnitude of the BCG-attributable increase in IFN-gamma responsiveness to M tuberculosis PPD, from before to 1 year post-vaccination, correlates better with the known levels of protection induced by immunisation with BCG than does the absolute value of the IFN-gamma or DTH response after vaccination. It is likely that differential sensitisation due to exposure to environmental mycobacteria is the most important determinant of the observed differences in protection by BCG between populations.

Expression of the developmental Sonic hedgehog (Shh) signalling pathway is up-regulated in chronic lung fibrosis and the Shh receptor patched 1 is present in circulating T lymphocytes

During pulmonary development, Sonic hedgehog (Shh) and transforming growth factor β1 (TGF‐β1) signalling both contribute to branching morphogenesis. In interstitial lung disease, the complex alveolar structure of the lung is disrupted and remodelled, which leads to fibrosis, loss of respiratory surface, morbidity, and mortality. It is well documented that TGF‐β1 is involved in fibrosis. However, little is known about Shh signalling in damaged epithelia. This study examined whether or not components of the Shh signalling pathway, as well as TGF‐β1, are expressed in human fibrotic lung disease (cryptogenic fibrosing alveolitis and bronchiectasis) and in murine experimental models of fibrotic and non‐fibrotic chronic pulmonary inflammation. Using immunohistochemistry, it was observed that Shh, like TGF‐β1, is up‐regulated in epithelial cells at sites of fibrotic disease but not non‐fibrotic inflammation. The Shh receptor patched was detected in infiltrating mononuclear cells and alveolar macrophages, as well as normal resting peripheral blood T lymphocytes. Neither Shh nor patched is expressed by hyperproliferative goblet cells in inflammatory epithelium. This study demonstrates that patched is present in human peripheral CD4 and CD8 lymphocytes at both protein and mRNA levels. Taken together, these results suggest that components of the highly conserved Shh signalling pathway may play a role in the remodelling of damaged pulmonary epithelium and that damaged epithelium and cells of the immune system may communicate via this pathway. Copyright

Suppression of allergen reactive Th2 mediated responses and pulmonary eosinophilia by intranasal administration of an immunodominant peptide is linked to IL-10 production

The potential to induce systemic tolerance following exposure of the airway mucosa to soluble antigen, may be applied therapeutically for the treatment of allergic disease. Since the use of allergen can trigger IgE mediated inflammation, we investigated whether mucosal delivery of a peptide, containing an immunodominant epitope of the Der p1 allergen of house dust mite, can lead to CD4(+) Th2 cell tolerance and thus protect against airway inflammatory responses to inhalant allergen. The administration of microencapsulated peptide to the nasal mucosa of mice, protected against airway inflammation, with significant reductions in eosinophil infiltration into the airways following allergen challenge. Der p1 specific antibody levels in sera were not modulated. Allergen reactive CD4(+) T cells expressed a tolerized phenotype, with reduction in levels of the cytokines, IL-5, IL-13 and IFN-gamma although IL-10 levels were increased. The mucosal administration of a peptide containing an immunodominant region of an allergen can protect against the induction of systemic and local inflammatory responses to allergen challenge.

Gamma Interferon Responses Induced by a Panel of Recombinant and Purified Mycobacterial Antigens in Healthy, Non-Mycobacterium bovis BCG-Vaccinated Malawian Young Adults

ABSTRACT We have previously shown that young adults living in a rural area of northern Malawi showed greater gamma interferon (IFN-γ) responses to purified protein derivatives (PPD) prepared from environmental mycobacteria than to PPD from Mycobacterium tuberculosis. In order to define the mycobacterial species to which individuals living in a rural African population have been exposed and sensitized, we tested T-cell recognition of recombinant and purified antigens from M. tuberculosis (38 kDa, MPT64, and ESAT-6), M. bovis (MPB70), M. bovis BCG (Ag85), and M. leprae (65 kDa, 35 kDa, and 18 kDa) in >600 non-M. bovis BCG-vaccinated young adults in the Karonga District of northern Malawi. IFN-γ was measured by enzyme-linked immunosorbent assay (ELISA) in day 6 supernatants of diluted whole-blood cultures. The recombinant M. leprae 35-kDa and 18-kDa and purified native M. bovis BCG Ag85 antigens induced the highest percentages of responders, though both leprosy and bovine tuberculosis are now rare in this population. The M. tuberculosis antigens ESAT-6 and MPT64 and the M. bovis antigen MPB70 induced the lowest percentages of responders. One of the subjects subsequently developed extrapulmonary tuberculosis; this individual had a 15-mm-diameter reaction to the Mantoux test and responded to M. tuberculosis PPD, Ag85, MPT64, and ESAT-6 but not to any of the leprosy antigens. We conclude that in this rural African population, exposure to M. tuberculosis or M. bovis is much less frequent than exposure to environmental mycobacteria such as M. avium, which have antigens homologous to the M. leprae 35-kDa and 18-kDa antigens. M. tuberculosis ESAT-6 showed the strongest association with the size of the Mantoux skin test induration, suggesting that among the three M. tuberculosis antigens tested it provided the best indication of exposure to, or infection with, M. tuberculosis.

Reversal of established CD4+ type 2 T helper-mediated allergic airway inflammation and eosinophilia by therapeutic treatment with DNA vaccines limits progression towards chronic inflammation and remodelling

Immunostimulatory DNA‐based vaccines can prevent the induction of CD4+ type 2 T helper (Th2) cell‐mediated airway inflammation in experimental models, when administered before or at the time of allergen exposure. Here we demonstrate their efficacy in limiting the progression of an established response to chronic pulmonary inflammation and airway remodelling on subsequent allergen challenge. Mice exhibiting Th2‐mediated airway inflammation induced following sensitization and challenge with group 1 allergen derived from Dermatophagoides pteronyssinus group species (Der p 1), a major allergen of house dust mite, were treated with pDNA vaccines. Their airways were rechallenged and the extent of inflammation assessed. In plasma DNA (pDNA)‐vaccinated mice, infiltration of inflammatory cells, goblet cell hyperplasia and mucus production were reduced and subepithelial fibrosis attenuated. The reduction in eosinophil numbers correlated with a fall in levels of the profibrotic mediator transforming growth factor (TGF)‐β1 in bronchoalveolar lavage (BAL) and lung tissue. In addition to lung epithelial cells and resident alveolar macrophages, infiltrating eosinophils, the principle inflammatory cells recruited following allergen exposure, were a major source of TGF‐β1. Protection, conferred irrespective of the specificity of the pDNA construct, did not correlate with a sustained increase in systemic interferon (IFN)‐γ production but in a reduction in levels of the Th2 pro‐inflammatory cytokines. Notably, there was a reduction in levels of interleukin (IL)‐5 and IL‐13 produced by systemic Der p 1 reactive CD4+ Th2 cells on in vitro stimulation as well as in IL‐4 and IL‐5 levels in BAL fluid. These data suggest that suppression of CD4+ Th2‐mediated inflammation and eosinophilia were sufficient to attenuate progression towards airway remodelling. Immunostimulatory DNA may therefore have a therapeutic application in treatment of established allergic asthma in patients.

Mycobacterial Purified Protein Derivatives Stimulate Innate Immunity: Malawians Show Enhanced Tumor Necrosis Factor Alpha, Interleukin-1β (IL-1β), and IL-10 Responses Compared to Those of Adolescents in the United Kingdom

ABSTRACT To investigate the role of innate immunity in variable efficacy of Mycobacterium bovis BCG vaccination in Malawi and the United Kingdom, we examined 24-h tumor necrosis factor alpha, interleukin-1β (IL-1β), and IL-10 responses to mycobacterial purified protein derivatives (PPDs). The rank order in stimulatory potency for different PPDs was the same for all three cytokines. Before vaccination Malawians made higher pro- and anti-inflammatory responses than did United Kingdom subjects. Fewer than 5% of United Kingdom subjects made IL-10 in response to any PPD, compared to 19 to 57% responders among Malawians. Priming for regulatory IL-10 may contribute to the smaller increase in gamma interferon responses in Malawians compared to United Kingdom subjects following BCG vaccination.

Reduced interleukin-8 response to Streptococcus pneumoniae by alveolar macrophages from adults with HIV/AIDS

Background:HIV-infected adults are highly susceptible to pneumococcal disease. Objective:To examine if alveolar macrophages from HIV-infected subjects exhibited a failure of cytokine production in response to Streptococcus pneumoniae in vitro. Design:Case–control comparison of alveolar macrophages from 11 HIV-infected and 13 non-infected adults. Methods:Type 1 opsonized S. pneumoniae were used to challenge the alveolar macrophages in vitro. Cell supernatant fluid was collected from unstimulated cells, and cells challenged with bacteria for 0, 6, 12 and 24 h. Cytokine production (interleukins 1β, 6 and 8) was measured in all fluids using an enzyme-linked immunosorbent assay. Results:All the cytokines tested increased over time in both HIV-infected and uninfected subjects. Interleukin-8 release was significantly lower in HIV-infected than in non-HIV-infected subjects (P = 0.02). Conclusion:Reduced interleukin-8 production may result in decreased neutrophil recruitment, and hence increased susceptibility to pneumococcal infection in HIV-infected subjects.

Inhaled delivery of 23-valent pneumococcal polysaccharide vaccine does not result in enhanced pulmonary mucosal immunoglobulin responses

We compared the effect of intramuscular vs. inhaled 23-valent pneumococcal capsular polysaccharide vaccine (23-PPV) on pulmonary mucosal immunoglobulin levels. Bronchoalveolar lavage (BAL) and serum were collected from 33 adults before and 1 month after injected (n = 16) or inhaled (n = 17) 23-PPV. Levels of pneumococcal capsule-specific IgG and IgA to types 1, 9V and 14 were measured in each sample. Injected 23-PPV produced a significant increase in types 1, 9V and 14 capsule-specific IgG and type 1 IgA in both serum and BAL (type 1 geometric mean BAL IgG 9.8 ng/ml post-vaccine vs. 5 ng/ml pre-vaccine, p = 0.01; type 9V geo mean 5.6 ng/ml vs. 2.7 ng/ml, p = 0.001; type 14 geo mean 23.6 ng/ml vs. 6.2 ng/ml, p = 0.02). Inhaled vaccine produced no response in either BAL or serum.

Different profile of CD8+ effector T cells induced in Der p 1‐allergic and naïve mice by DNA vaccination

DNA vaccination holds great promise in both prophylactic and therapeutic vaccines. Recent evidence suggests that DNA vaccines could be powerful therapies countering Th2‐mediated disorders suchas allergies. Here, we studied the allergen‐specific CD4+ and CD8+ T cell populations induced following immunization of allergic and non‐allergic mice with DNA vaccine vectors encoding discrete epitopes of the house dust mite (HDM) Dermatophagoides pteronyssinus group I (Der p 1) allergen. Specifically, mice were sensitized to Der p 1 and exhibited a strong Th2/allergic response. Sensitized and non‐allergic mice were then compared for their responses to DNA immunization. Using Elispot analysis, we demonstrate that allergic/vaccinated mice generate a mixed Th1/Th2 response against the allergen with high numbers of allergen‐specific CD4+ T cells secreting IFN‐γ or IL‐4, whereas in non‐allergic/vaccinated mice a polarized Th1 response was dominant. Allergen‐specific CD8+ T cells secreting IFN‐γ were induced at equal frequencies in both allergic and non‐allergic mice. However, the CD8+ T cells from allergic mice were markedly deficient in their cytotoxic potential when compared to their counterparts in non‐allergic mice. These results indicate that during an ongoing Th2 response, DNA vaccination leads to the generation of a distinct population of non‐cytotoxic/regulatory CD8+ T cells.

The alveolar microenvironment of patients infected with human immunodeficiency virus does not modify alveolar macrophage interactions with Streptococcus pneumoniae.

ABSTRACT We tested the hypothesis that HIV infection results in activation of alveolar macrophages and that this might be associated with impaired defense against pneumococcus. We compared alveolar macrophages and lymphocytes in 131 bronchoalveolar lavage samples from HIV-infected and healthy controls using inflammatory gene microarrays, flow cytometry, real-time PCR, and enzyme-linked immunosorbent assay (ELISA) to determine the pattern of macrophage activation associated with HIV infection and the effect of this activation on defense against pneumococcus. We used gamma interferon (IFN-γ) priming to mimic the cellular milieu in HIV-infected lungs. InnateDB and BioLayout 3D were used to analyze the interactions of the upregulated genes. Alveolar macrophages from HIV-infected adults showed increased gene expression and cytokine production in a classical pattern. Bronchoalveolar lavage from HIV-infected subjects showed excess CD8+ lymphocytes with activated phenotype. Toll-like receptor 4 (TLR4) expression was increased in macrophages from HIV-infected subjects, but function was similar between the groups; lung lavage fluid did not inhibit TLR function in transfected HeLa cells. Alveolar macrophages from HIV-infected subjects showed normal binding and internalization of opsonized pneumococci, with or without IFN-γ priming. Alveolar macrophages from HIV-infected subjects showed classical activation compared to that of healthy controls, but this does not alter macrophage interactions with pneumococci.