A novel lung explant model for the ex vivo study of efficacy and mechanisms of anti-influenza drugs.

Influenza A virus causes considerable morbidity and mortality largely because of a lack of effective antiviral drugs. Viral neuraminidase inhibitors, which inhibit viral release from the infected cell, are currently the only approved drugs for influenza, but have recently been shown to be less effective than previously thought. Growing resistance to therapies that target viral proteins has led to increased urgency in the search for novel anti-influenza compounds. However, discovery and development of new drugs have been restricted because of differences in susceptibility to influenza between animal models and humans and a lack of translation between cell culture and in vivo measures of efficacy. To circumvent these limitations, we developed an experimental approach based on ex vivo infection of human bronchial tissue explants and optimized a method of flow cytometric analysis to directly quantify infection rates in bronchial epithelial tissues. This allowed testing of the effectiveness of TVB024, a vATPase inhibitor that inhibits viral replication rather than virus release, and to compare efficacy with the current frontline neuraminidase inhibitor, oseltamivir. The study showed that the vATPase inhibitor completely abrogated epithelial cell infection, virus shedding, and the associated induction of proinflammatory mediators, whereas oseltamivir was only partially effective at reducing these mediators and ineffective against innate responses. We propose, therefore, that this explant model could be used to predict the efficacy of novel anti-influenza compounds targeting diverse stages of the viral replication cycle, thereby complementing animal models and facilitating progression of new drugs into clinical trials.

Toll-like Receptor 7 Is Reduced in Severe Asthma and Linked to an Altered MicroRNA Profile.

RATIONALE: Asthma is one of the most common chronic diseases worldwide, and individuals with severe asthma experience recurrent exacerbations. Exacerbations are predominantly viral associated and have been linked to defective airway IFN responses. Ascertaining the molecular mechanisms underlying this deficiency is a major research goal to identify new therapeutic targets. OBJECTIVES: We investigated the hypothesis that reduced Toll-like receptor 7 (TLR7)-derived signaling drove the impaired IFN responses to rhinovirus by asthmatic alveolar macrophages (AMs); the molecular mechanisms underlying this deficiency were explored. METHODS: AMs were recovered from bronchoalveolar lavage from healthy subjects and patients with severe asthma. Expression of pattern-recognition receptors and microRNAs was evaluated by quantitative polymerase chain reaction and Western blotting. A TLR7-luciferase reporter construct was created to evaluate binding of microRNAs to the 3' untranslated region of TLR7. IFN production was measured by quantitative polymerase chain reaction and ELISA. MEASUREMENTS AND MAIN RESULTS: The expression of TLR7 was significantly reduced in severe asthma AMs and was associated with reduced rhinovirus and imiquimod-induced IFN responses by these cells compared with healthy AMs. Severe asthma AMs also expressed increased levels of three microRNAs, which we showed were able to directly reduce TLR7 expression. Ex vivo knockdown of these microRNAs restored TLR7 expression with concomitant augmentation of virus-induced IFN production. CONCLUSIONS: In severe asthma, TLR7 deficiency drives impaired innate immune responses to virus by AMs. Blocking a group of microRNAs that are up-regulated in these cells can restore antiviral innate responses, providing a novel approach for therapy in asthma.

Evaluation of the efficacy of ChAd63-MVA vectored vaccines expressing circumsporozoite protein and ME-TRAP against controlled human malaria infection in malaria-naive individuals.

BACKGROUND: Circumsporozoite protein (CS) is the antigenic target for RTS,S, the most advanced malaria vaccine to date. Heterologous prime-boost with the viral vectors simian adenovirus 63 (ChAd63)-modified vaccinia virus Ankara (MVA) is the most potent inducer of T-cells in humans, demonstrating significant efficacy when expressing the preerythrocytic antigen insert multiple epitope-thrombospondin-related adhesion protein (ME-TRAP). We hypothesized that ChAd63-MVA containing CS may result in a significant clinical protective efficacy. METHODS: We conducted an open-label, 2-site, partially randomized Plasmodium falciparum sporozoite controlled human malaria infection (CHMI) study to compare the clinical efficacy of ChAd63-MVA CS with ChAd63-MVA ME-TRAP. RESULTS: One of 15 vaccinees (7%) receiving ChAd63-MVA CS and 2 of 15 (13%) receiving ChAd63-MVA ME-TRAP achieved sterile protection after CHMI. Three of 15 vaccinees (20%) receiving ChAd63-MVA CS and 5 of 15 (33%) receiving ChAd63-MVA ME-TRAP demonstrated a delay in time to treatment, compared with unvaccinated controls. In quantitative polymerase chain reaction analyses, ChAd63-MVA CS was estimated to reduce the liver parasite burden by 69%-79%, compared with 79%-84% for ChAd63-MVA ME-TRAP. CONCLUSIONS: ChAd63-MVA CS does reduce the liver parasite burden, but ChAd63-MVA ME-TRAP remains the most promising antigenic insert for a vectored liver-stage vaccine. Detailed analyses of parasite kinetics may allow detection of smaller but biologically important differences in vaccine efficacy that can influence future vaccine development. CLINICAL TRIALS REGISTRATION: NCT01623557.

Effect of a quadrivalent meningococcal ACWY glycoconjugate or a serogroup B meningococcal vaccine on meningococcal carriage: an observer-blind, phase 3 randomised clinical trial.

BACKGROUND: Meningococcal conjugate vaccines protect individuals directly, but can also confer herd protection by interrupting carriage transmission. We assessed the effects of meningococcal quadrivalent glycoconjugate (MenACWY-CRM) or serogroup B (4CMenB) vaccination on meningococcal carriage rates in 18-24-year-olds. METHODS: In this phase 3, observer-blind, randomised controlled trial, university students aged 18-24 years from ten sites in England were randomly assigned (1:1:1, block size of three) to receive two doses 1 month apart of Japanese Encephalitis vaccine (controls), 4CMenB, or one dose of MenACWY-CRM then placebo. Participants were randomised with a validated computer-generated random allocation list. Participants and outcome-assessors were masked to the treatment group. Meningococci were isolated from oropharyngeal swabs collected before vaccination and at five scheduled intervals over 1 year. Primary outcomes were cross-sectional carriage 1 month after each vaccine course. Secondary outcomes included comparisons of carriage at any timepoint after primary analysis until study termination. Reactogenicity and adverse events were monitored throughout the study. Analysis was done on the modified intention-to-treat population, which included all enrolled participants who received a study vaccination and provided at least one assessable swab after baseline. This trial is registered with ClinicalTrials.gov, registration number NCT01214850. FINDINGS: Between Sept 21 and Dec 21, 2010, 2954 participants were randomly assigned (987 assigned to control [984 analysed], 979 assigned to 4CMenB [974 analysed], 988 assigned to MenACWY-CRM [983 analysed]); 33% of the 4CMenB group, 34% of the MenACWY-CRM group, and 31% of the control group were positive for meningococcal carriage at study entry. By 1 month, there was no significant difference in carriage between controls and 4CMenB (odds ratio 1·2, 95% CI 0·8-1·7) or MenACWY-CRM (0·9, [0·6-1·3]) groups. From 3 months after dose two, 4CMenB vaccination resulted in significantly lower carriage of any meningococcal strain (18·2% [95% CI 3·4-30·8] carriage reduction), capsular groups BCWY (26·6% [10·5-39·9] carriage reduction), capsular groups CWY (29·6% [8·1-46·0] carriage reduction), and serogroups CWY (28·5% [2·8-47·5] carriage reduction) compared with control vaccination. Significantly lower carriage rates were also noted in the MenACWY-CRM group compared with controls: 39·0% (95% CI 17·3-55·0) carriage reduction for serogroup Y and 36·2% (15·6-51·7) carriage reduction for serogroup CWY. Study vaccines were generally well tolerated, with increased rates of transient local injection pain and myalgia in the 4CMenB group. No safety concerns were identified. INTERPRETATION: Although we detected no significant difference between groups at 1 month after vaccine course, MenACWY-CRM and 4CMenB vaccines reduced meningococcal carriage rates during 12 months after vaccination and therefore might affect transmission when widely implemented. FUNDING: Novartis Vaccines.

ChAd63-MVA-vectored blood-stage malaria vaccines targeting MSP1 and AMA1: assessment of efficacy against mosquito bite challenge in humans.

The induction of cellular immunity, in conjunction with antibodies, may be essential for vaccines to protect against blood-stage infection with the human malaria parasite Plasmodium falciparum. We have shown that prime-boost delivery of P. falciparum blood-stage antigens by chimpanzee adenovirus 63 (ChAd63) followed by the attenuated orthopoxvirus MVA is safe and immunogenic in healthy adults. Here, we report on vaccine efficacy against controlled human malaria infection delivered by mosquito bites. The blood-stage malaria vaccines were administered alone, or together (MSP1+AMA1), or with a pre-erythrocytic malaria vaccine candidate (MSP1+ME-TRAP). In this first human use of coadministered ChAd63-MVA regimes, we demonstrate immune interference whereby responses against merozoite surface protein 1 (MSP1) are dominant over apical membrane antigen 1 (AMA1) and ME-TRAP. We also show that induction of strong cellular immunity against MSP1 and AMA1 is safe, but does not impact on parasite growth rates in the blood. In a subset of vaccinated volunteers, a delay in time to diagnosis was observed and sterilizing protection was observed in one volunteer coimmunized with MSP1+AMA1-results consistent with vaccine-induced pre-erythrocytic, rather than blood-stage, immunity. These data call into question the utility of T cell-inducing blood-stage malaria vaccines and suggest that the focus should remain on high-titer antibody induction against susceptible antigen targets.

Preliminary assessment of the efficacy of a T-cell-based influenza vaccine, MVA-NP+M1, in humans.

BACKGROUND: The novel influenza vaccine MVA-NP+M1 is designed to boost cross-reactive T-cell responses to internal antigens of the influenza A virus that are conserved across all subtypes, providing protection against both influenza disease and virus shedding against all influenza A viruses. Following a phase 1 clinical study that demonstrated vaccine safety and immunogenicity, a phase 2a vaccination and influenza challenge study has been conducted in healthy adult volunteers. METHODS: Volunteers with no measurable serum antibodies to influenza A/Wisconsin/67/2005 received either a single vaccination with MVA-NP+M1 or no vaccination. T-cell responses to the vaccine antigens were measured at enrollment and again prior to virus challenge. All volunteers underwent intranasal administration of influenza A/Wisconsin/67/2005 while in a quarantine unit and were monitored for symptoms of influenza disease and virus shedding. RESULTS: Volunteers had a significantly increased T-cell response to the vaccine antigens following a single dose of the vaccine, with an increase in cytolytic effector molecules. Intranasal influenza challenge was undertaken without safety issues. Two of 11 vaccinees and 5 of 11 control subjects developed laboratory-confirmed influenza (symptoms plus virus shedding). Symptoms of influenza were less pronounced in the vaccinees and there was a significant reduction in the number of days of virus shedding in those vaccinees who developed influenza (mean, 1.09 days in controls, 0.45 days in vaccinees, P = .036). CONCLUSIONS: This study provides the first demonstration of clinical efficacy of a T-cell-based influenza vaccine and indicates that further clinical development should be undertaken. CLINICAL TRIALS REGISTRATION: NCT00993083.

Optimisation of COVID-19 diagnostic pathways in acute hospital admissions to prevent nosocomial transmission.

INTRODUCTION: In the management of acute hospital admissions during the COVID-19 pandemic, safe patient cohorting depends on robust admission diagnostic strategies. It is essential that screening strategies are sensitive and rapid, to prevent nosocomial transmission of COVID-19 and maintain patient flow. METHODS: We retrospectively identified all COVID-19 positive and suspected cases at our institution screened by reverse transcription polymerase chain reaction (RT-PCR) between 4 April and 28 June 2020. Using RT-PCR positivity within 7 days as our reference standard, we assessed sensitivity and net-benefit of three admission screening strategies: single admission RT-PCR, composite admission RT-PCR and CXR and repeat RT-PCR with 48 h. RESULTS: RT-PCR single-test sensitivity was 91.5% (87.8%-94.4%) versus 97.7% (95.4%-99.1%) (p = 0.025) for RT-PCR/CXR composite testing and 95.1% (92.1%-97.2%) (p = 0.03) for repeated RT-PCR. Net-benefit was 0.83 for single RT-PCR versus 0.89 for RT-PCR/CXR and 0.87 for repeated RT-PCR at 0.02% threshold probability. CONCLUSION: The RT-PCR/CXR composite testing strategy was highly sensitive when screening patients at the point of hospital admission. Real-world sensitivity of this approach was comparable to repeat RT-PCR testing within 48 h; however, faster facilitating improved patient flow.

A general framework in single and multi-modality registration for lung imaging analysis using statistical prior shapes.

BACKGROUND AND OBJECTIVE: A fusion of multi-slice computed tomography (MSCT) and single photon emission computed tomography (SPECT) represents a powerful tool for chronic obstructive pulmonary disease (COPD) analysis. In this paper, a novel and high-performance MSCT/SPECT non-rigid registration algorithm is proposed to accurately map the lung lobe information onto the functional imaging. Such a fusion can then be used to guide lung volume reduction surgery. METHODS: The multi-modality fusion method proposed here is developed by a multi-channel technique which performs registration from MSCT scan to ventilation and perfusion SPECT scans simultaneously. Furthermore, a novel function with less parameters is also proposed to avoid the adjustment of the weighting parameter and to achieve a better performance in comparison with the exisitng methods in the literature. RESULTS: A lung imaging dataset from a hospital and a synthetic dataset created by software are employed to validate single- and multi-modality registration results. Our method is demonstrated to achieve the improvements in terms of registration accuracy and stability by up to 23% and 54% respectively. Our multi-channel technique proposed here is also proved to obtain improved registration accuracy in comparison with single-channel method. CONCLUSIONS: The fusion of lung lobes onto SPECT imaging is achievable by accurate MSCT/SPECT alignment. It can also be used to perform lobar lung activity analysis for COPD diagnosis and treatment.

Assessment of novel vaccination regimens using viral vectored liver stage malaria vaccines encoding ME-TRAP.

Heterologous prime-boost vaccination with viral vectors simian adenovirus 63 (ChAd63) and Modified Vaccinia Ankara (MVA) induces potent T cell and antibody responses in humans. The 8-week regimen demonstrates significant efficacy against malaria when expressing the pre-erythrocytic malaria antigen Thrombospondin-Related Adhesion Protein fused to a multiple epitope string (ME-TRAP). We tested these vaccines in 7 new 4- and 8- week interval schedules to evaluate safety and immunogenicity of multiple ChAd63 ME-TRAP priming vaccinations (denoted A), multiple MVA ME-TRAP boosts (denoted M) and alternating vectors. All regimens exhibited acceptable reactogenicity and CD8+ T cell immunogenicity was enhanced with a 4-week interval (AM) and with incorporation of additional ChAd63 ME-TRAP vaccination at 4- or 8-weeks (AAM or A_A_M). Induction of TRAP antibodies was comparable between schedules. T cell immunity against the ChAd63 hexon did not affect T cell responses to the vaccine insert, however pre-vaccination ChAd63-specific T cells correlated with reduced TRAP antibodies. Vaccine-induced antibodies against MVA did not affect TRAP antibody induction, and correlated positively with ME-TRAP-specific T cells. This study identifies potentially more effective immunisation regimens to assess in Phase IIa trials and demonstrates a degree of flexibility with the timing of vectored vaccine administration, aiding incorporation into existing vaccination programmes.

The histone deacetylase inhibitor, romidepsin, as a potential treatment for pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease that usually affects elderly people. It has a poor prognosis and there are limited therapies. Since epigenetic alterations are associated with IPF, histone deacetylase (HDAC) inhibitors offer a novel therapeutic strategy to address the unmet medical need. This study investigated the potential of romidepsin, an FDA-approved HDAC inhibitor, as an anti-fibrotic treatment and evaluated biomarkers of target engagement that may have utility in future clinical trials. The anti-fibrotic effects of romidepsin were evaluated both in vitro and in vivo together with any harmful effect on alveolar type II cells (ATII). Bronchoalveolar lavage fluid (BALF) from IPF or control donors was analyzed for the presence of lysyl oxidase (LOX). In parallel with an increase in histone acetylation, romidepsin potently inhibited fibroblast proliferation, myofibroblast differentiation and LOX expression. ATII cell numbers and their lamellar bodies were unaffected. In vivo, romidepsin inhibited bleomycin-induced pulmonary fibrosis in association with suppression of LOX expression. LOX was significantly elevated in BALF of IPF patients compared to controls. These data show the anti-fibrotic effects of romidepsin, supporting its potential use as novel treatment for IPF with LOX as a companion biomarker for evaluation of early on-target effects.

The Relationship Between Crackle Characteristics and Airway Morphology in COPD.

BACKGROUND: Crackles in COPD are believed to be generated by the re-opening of collapsed airways, which result from chronic inflammation, secretions, and loss of cartilaginous support through inflammation. However, it is unclear whether crackle characteristics can be used to identify COPD. This is the first study to examine the relationship between specific added lung sounds (crackles) and measurements of conductive airways and emphysema score obtained from high-resolution computed tomography (HRCT) in vivo in humans. A predictive relationship might permit the use of lung sounds as a biomarker for COPD. METHODS: A convenience sample of 26 subjects was recruited into the study and consisted of 9 healthy non-smokers, 9 healthy smokers, and 8 subjects with mild or moderate COPD. Lung sound data were recorded using a digital stethoscope connected to a laptop computer. Airway diameter, emphysema score, and percentage of wall area were measured from HRCT scans. RESULTS: The analysis showed that there were no statistically significant differences in crackle characteristics (the number of crackles per breathing cycle and crackle 2-cycle duration) between the 3 subject groups. Both crackle 2-cycle duration and crackle number showed some significant correlation with airway parameters at some branch generations, but due to the large number of correlations performed, these were consistent with chance findings. CONCLUSIONS: Although there were some significant correlations between crackle characteristics and measurements of the conductive airways and emphysema score, the possibility that these correlations have occurred by chance cannot be ruled out. Therefore, this study provides no conclusive evidence that crackle characteristics are related to HRCT variables in COPD.

Regional assessment of lung function using thin-plate splines to align structural and functional imaging.

Ventilation / perfusion (VQ) Single Photon Emission Computed Tomography (SPECT) imaging provides 3D data of the regional distribution of ventilation and perfusion throughout the lung, but interpretation of the results is difficult without reference to the underlying lung anatomy. Multi-Slice Computed Tomography (MSCT) imaging is able to provide significant anatomical detail in the lung, allowing delineation of regional features such as the lobes. The purpose of this work was to develop software tools to allow the alignment of regions delineated from the MSCT scans, with the corresponding SPECT data, to allow measurements of VQ to be made for anatomically meaningful regions. The technique developed was based on the use of thin-plate splines and the results showed that it was able to provide good alignment between the MSCT and SPECT data.

The co-imaging of gamma camera measurements of aerosol deposition and respiratory anatomy.

The use of gamma camera imaging following the inhalation of a radiolabel has been widely used by researchers to investigate the fate of inhaled aerosols. The application of two-dimensional (2D) planar gamma scintigraphy and single-photon emission computed tomography (SPECT) to the study of inhaled aerosols is discussed in this review. Information on co-localized anatomy can be derived from other imaging techniques such as krypton ventilation scans and low- and high-resolution X-ray computed tomography (CT). Radionuclide imaging, combined with information on anatomy, is a potentially useful approach when the understanding of regional deposition within the lung is central to research objectives for following disease progression and for the evaluation of therapeutic intervention.