Holly D. Clifford

Toll-like receptor 7 and 8 polymorphisms: associations with functional effects and cellular and antibody responses to measles virus and vaccine

Successful defence against viral pathogens requires the rapid recognition of virus-specific “danger signals” and the activation of both innate and adaptive immunity. Toll-like receptors (TLR) 7 and 8 play a critical role in the elimination of viruses by recognising the common viral component, single stranded (ss)RNA. Measles virus, an ssRNA virus, continues to cause serious morbidity and mortality worldwide despite available measles vaccines. TLR7 and TLR8 genetic variation may cause functional alterations that result in impaired responses to measles. In a population of 12-month-old Australian infants, receptor protein expression was examined to assess the functionality of TLR7 and TLR8 polymorphisms, and the effects of these polymorphisms on cellular and antibody responses after the first measles vaccine dose were investigated. TLR7 Leu11Gln showed associations with TNF-α responses after ligand (imiquimod) stimulation in males only (P = 0.040), and non-responders were more likely to be Gln males (P = 0.044). TNF-α non-responders after imiquimod also had higher percentages of TLR8 -4284TT (69.6%) (P = 0.001) and TLR8 -558CC (69.6%) (P = 0.002) in females. Receptor protein expression after imiquimod or measles stimulation was not significantly altered compared with baseline, nor was it affected by genotype. None of the TLR7 or TLR8 polymorphisms studied were associated with measles-specific cytokine levels or with measles IgG levels. In conclusion, we report gender-specific associations with TLR7 and TLR8 polymorphisms and TNF-α cellular responses to its ligand. However, we found no evidence of any functional effects of TLR7 or TLR8 polymorphisms on receptor expression, measles-specific cellular responses or measles vaccine antibody responses.

Polymorphisms in key innate immune genes and their effects on measles vaccine responses and vaccine failure in children from Mozambique

Despite an effective vaccine, measles remains a major health problem globally, particularly in developing countries. More than 30% of children show primary vaccine failure and therefore remain vulnerable to measles. Genetic variation in key innate pathogen recognition receptors, such as the measles cell entry receptors CD46 and SLAM, measles attachment receptor DC-SIGN, the antiviral toll-like receptors (TLR)3, TLR7 and TLR8, and the cytosolic antiviral receptor RIG-I, may significantly affect measles IgG antibody responses. Measles is still highly prevalent in developing countries such as those in Africa however there is no previous data on the effect of these innate immune genes in a resident African population. Polymorphisms (n=29) in the candidate genes were genotyped in a cohort of vaccinated children (n=238) aged 6 months-14 years from Mozambique, Africa who either had vaccine failure and contracted measles (cases; n=66) or controls (n=172). Contrasting previous associations with measles responses in Caucasians and/or strong evidence for candidacy, we found little indication that these key innate immune genes affect measles IgG responses in our cohort of Mozambican children. We did however identify that CD46 and TLR8 variants may be involved in the occurrence of measles vaccine failure. This study highlights the importance of genetic studies in resident, non-Caucasian populations, from areas where determining the factors that may affect measles control is of a high priority.

TLR3 and RIG-I gene variants: Associations with functional effects on receptor expression and responses to measles virus and vaccine in vaccinated infants

Measles virus causes severe morbidity and mortality, despite the availability of measles vaccines. Successful defence against viral pathogens requires early recognition of virus-specific patterns by innate receptors like Toll-like receptor (TLR)3 and the RNA helicase, retinoic acid inducible gene-I (RIG-I). Genetic differences in these receptors may influence the primary immune responses to measles and the efficacy of measles vaccine. In 1-year-old Australian infants after their first measles vaccine dose, we investigated functional effects of TLR3 and RIG-I polymorphisms on intracellular protein expression using flow cytometry, cytokine responses to receptor ligands and measles lysate, and post-vaccination measles IgG levels. We found that TLR3 Leu412Phe was significantly associated with IFN-α/β response after stimulation with TLR3 ligand, poly(I:C) (P=0.024). Downregulation of TLR3 protein expression in NK cells after poly(I:C) was also associated with this variant (P=0.011). In contrast, measles-specific expression, cytokine responses and antibody responses were not associated with TLR3 polymorphisms. No associations were found with RIG-I variants. These results suggest that a TLR3 polymorphism has functional effects on receptor expression and cytokine response. However, this did not translate to an effect on specific responses to measles virus or vaccine. We found no evidence that RIG-I polymorphisms were involved in measles immune responses.

Slam and dc-sign measles receptor polymorphisms and their impact on antibody and cytokine responses to measles vaccine

BACKGROUND Despite the use of measles vaccine, measles virus continues to circulate and cause severe disease. Immune responses to the measles vaccine are variable between individuals, with up to 10% failing to produce a sufficient protective response post-vaccination. Signaling lymphocyte activation molecule (SLAM) and dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN; CD209) are specific measles receptors: SLAM binds and permits entry of the virus into the cell, DC-SIGN acts as an attachment receptor, increasing viral binding efficiency and transmission. Genetic variations in these receptor genes may alter measles vaccine antibody and cellular responses. METHODS In 12-month-old infants from Perth, Western Australia after their first measles vaccine dose as part of the combination measles-mumps-rubella (MMR) vaccine, 7 SLAM and DC-SIGN polymorphisms were genotyped and associations were investigated with measles IgG antibody levels and in vitro measles cytokine responses. RESULTS The DC-SIGN promoter variant -336C/T was associated with overall IFN-γ responses after measles stimulation (P=0.002) and three DC-SIGN polymorphisms (-336C/T, -139C/T and -871C/T) were associated with the proportion of cytokine non-responders to measles (P=0.001, P=0.021 and P=0.036, respectively). However, no associations were found between the DC-SIGN or SLAM polymorphisms and measles IgG antibody levels. CONCLUSIONS The results suggest that DC-SIGN -139C/T, -336C/T and -871C/T polymorphisms may modulate cytokine (but not antibody) responses to the measles component of MMR vaccine. Furthermore, contrasting previous studies, SLAM polymorphisms do not appear to affect measles antibody or cytokine responses in this cohort.

CD46 Measles Virus Receptor Polymorphisms Influence Receptor Protein Expression and Primary Measles Vaccine Responses in Naive Australian Children

ABSTRACT Despite the availability of measles vaccines, infants continue to die from measles. Measles vaccine responses vary between individuals, and poor immunogenicity is likely to preclude protection against measles. CD46 is a ubiquitously expressed specific receptor for vaccine strains of measles virus. CD46 polymorphisms have not been functionally investigated but may affect CD46 protein expression, which in turn may mediate primary measles antibody responses in infants. In a cohort of children aged 12 to 14 months from Perth, Australia (n = 137), after their first dose of measles-mumps-rubella (MMR) vaccine, CD46 polymorphisms were genotyped, and postvaccination measles IgG and CD46 protein expression before and after measles lysate stimulation of cells were measured. Three CD46 variants (rs7144, rs11118580, and rs2724384) were significantly associated with measles virus-specific IgG levels (P = 0.008, P = 0.026, and P = 0.018, respectively). There were significant differences between CD46 rs7144 genotypes and CD46 protein expression on T cells, as well as the downregulation of CD46 and T-cell frequency after measles lysate stimulation. We show that CD46 polymorphisms were associated with primary measles antibody responses in naive infants. We also report the first association of a measles virus receptor polymorphism with functional effects on the receptor, suggesting a possible mechanism through which antibody responses are altered. Elucidating all of the interconnecting genetic factors that alter primary measles vaccine responses may be important for identifying children at risk of poor immunogenicity or vaccine failure and for the future design of vaccine strategies to help these children.

Geogenic PM10 exposure exacerbates responses to influenza infection

Particulate matter (PM) exposure has been linked epidemiologically to exacerbations of lung disease, including respiratory infections. We investigated the effects of geogenic (earth-derived) PM10 (PM<10 μm diameter) on the response to a respiratory viral infection. Geogenic dust was sampled from four communities in arid environments in Western Australia. Adult female BALB/c mice were intranasally exposed to chronic doses of PM10 (10 μg/day for 10 days), and/or infected with influenza (A/Mem/1/71) virus. Inflammation (cells, IL-6, IFN-γ) was measured in bronchoalveolar lavage. Lung mechanics were measured using the forced oscillation technique. Geogenic PM10 induced lung inflammation (neutrophils, macrophages) with additive effects in mice also infected with influenza. PM10 also modified the influenza-induced IL-6 and IFN-γ responses. Geogenic PM10 increased airway resistance, and increased hysteresivity in those exposed to both insults. Viral titres were significantly higher after PM10 exposure. Iron concentration was inversely associated with IFN-γ and positively associated with viral titre and hysteresivity. Geogenic PM10 exposure increases inflammation, impairs lung function and increases viral load, exacerbating the response to respiratory viral infection. Iron in the particles may be a driver of these responses. This has important implications for respiratory health in communities exposed to high geogenic PM10, such as those in arid environments.

A cross-sectional survey of environmental health in remote Aboriginal communities in Western Australia.

Abstract Introduction: The Australian Aboriginal population experiences significantly poorer health than the non-Aboriginal population. The contribution of environmental risk factors in remote communities to this health disparity is poorly understood. Objective: To describe and quantify major environmental risk factors and associated health outcomes in remote Aboriginal communities in Western Australia. Methods: The association between environmental health indicators, community infrastructure and reported health outcomes was analysed using linear and logistic regression of survey data. Results: Housing/overcrowding was significantly associated with increased reports of hearing/eyesight (OR 3.01 95 % CI 1.58–5.73), skin (OR 2.71 95 % CI 1.31–5.60), gastrointestinal (OR 3.51 95 % CI 1.49–8.26) and flu/colds (OR 2.47 95 % CI 1.27–4.78) as health concerns. Dust was significantly associated with hearing/eyesight (OR 3.16 95 % CI 1.82–5.48), asthma/respiratory (OR 2.48 95 % CI 1.43–4.29) and flu/colds (OR 3.31 95 % CI 1.88–5.86) as health concerns. Conclusion: Poor environmental health is prevalent in remote Aboriginal communities and requires further delineation to inform environmental health policy.

Bacillus licheniformis in geogenic dust induces inflammation in respiratory epithelium

&NA; Exposure to environmental geogenic (or earth‐derived) dust can lead to more frequent and severe infections in the human airway. Particulate matter < 10 &mgr;m (PM10) is the component of air pollution that is commonly associated with the exacerbation of respiratory diseases. We have previously demonstrated that mice exposed to geogenic dust PM10 experienced an exacerbation of inflammatory responses to influenza A virus. Whether geogenic dust PM10 also exacerbates respiratory bacterial infection is not yet known, nor are the components of the dust that drive these responses. We treated airway bronchial epithelial cells (NuLi‐1) with UV‐irradiated geogenic dust PM10 from six remote Western Australian towns. High levels of IL‐6 and IL‐8 production were observed, as well as persistent microbial growth. 16 S rRNA sequencing of the growth identified the microbe as Bacillus licheniformis, a spore‐forming, environmentally abundant bacterium. We next investigated the interaction of B. licheniformis with respiratory epithelium in vitro to determine whether this exacerbated infection with a bacterial respiratory pathogen (non‐typeable Haemophilus influenzae, NTHi). Heat treatment (100 °C) of all PM10 samples eliminated B. licheniformis contamination and reduced epithelial inflammatory responses, suggesting that heat‐labile and/or microbial factors were involved in the host response to geogenic dust PM10. We then exposed NuLi‐1 epithelium to increasing doses of the isolated Bacillus licheniformis (multiplicity of infection of 10:1, 1:1 or 0.1:1 bacteria: cells) for 1, 3, and 24 h. B. licheniformis and NTHi infection (association and invasion) was assessed using a standard gentamicin survival assay, and epithelial release of IL‐6 and IL‐8 was measured using a bead based immunoassay. B. licheniformis was cytotoxic to NuLi‐1 cells at 24 h. At 3 h post‐challenge, B. licheniformis elicited high IL‐6 and IL‐8 inflammatory responses from NuLi‐1 cells compared with cells treated with heat‐treated geogenic dust PM10 (p < 0.0001). Whilst treatment of cells with B. licheniformis increased inflammation, this did not make the cells more susceptible to NTHi infection. This study highlights that geogenic dust PM10 can harbour viable bacterial spores that induce inflammation in respiratory epithelium. The impact on respiratory health from inhalation of bacterial spores in PM10 in arid environments may be underestimated. Further investigation into the contribution of B. licheniformis and the wider dust microbiome to respiratory infection is warranted. HighlightsEarth‐derived geogenic dust exposure can lead to infections in the human airway.Spore‐forming Bacillus licheniformis is present in rural geogenic PM10.Heat‐treating geogenic dust removes its stimulatory effects on airway epithelium.High airway IL‐6 and IL‐8 levels are induced by live Bacillus licheniformis.Bacillus licheniformis may exacerbate infections associated with dust exposure.