Innate-like Gene Expression of Lung-Resident Memory CD8+ T Cells during Experimental Human Influenza: A Clinical Study.

Rationale: Suboptimal vaccine immunogenicity and antigenic mismatch, compounded by poor uptake, means that influenza remains a major global disease. T cells recognizing peptides derived from conserved viral proteins could enhance vaccine-induced cross-strain protection. Objectives: To investigate the kinetics, phenotypes, and function of influenza virus-specific CD8+ resident memory T (Trm) cells in the lower airway and infer the molecular pathways associated with their response to infection in vivo. Methods: Healthy volunteers, aged 18-55, were inoculated intranasally with influenza A/California/4/09(H1N1). Blood, upper airway, and (in a subgroup) lower airway samples were obtained throughout infection. Symptoms were assessed by using self-reported diaries, and the nasal viral load was assessed by using quantitative PCR. T-cell responses were analyzed by using a three-color FluoroSpot assay, flow cytometry with MHC I-peptide tetramers, and RNA sequencing, with candidate markers being confirmed by using the immunohistochemistry results for endobronchial biopsy specimens. Measurements and Main Results: After challenge, 57% of participants became infected. Preexisting influenza-specific CD8+ T cells in blood correlated strongly with a reduced viral load, which peaked at Day 3. Influenza-specific CD8+ T cells in BAL fluid were highly enriched and predominantly expressed the Trm markers CD69 and CD103. Comparison between preinfection CD8+ T cells in BAL fluid and blood by using RNA sequencing revealed 3,928 differentially expressed genes, including all major Trm-cell markers. However, gene set enrichment analysis of BAL-fluid CD8+ T cells showed primarily innate cell-related pathways and, during infection, included upregulation of innate chemokines (Cxcl1, Cxcl10, and Cxcl16) that were also expressed by CD8+ cells in bronchial tissues. Conclusions: CD8+ Trm cells in the human lung display innate-like gene and protein expression that demonstrates blurred divisions between innate and adaptive immunity. Clinical study registered with www.clinicaltrials.gov (NCT02755948).

Visualisation of direct interaction of MDA5 and the dsRNA replicative intermediate form of positive strand RNA viruses.

The innate immune system is a vital part of the body's defences against viral pathogens. The proteins retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation associated gene 5 (MDA5) function as cytoplasmic pattern recognition receptors that are involved in the elimination of actively replicating RNA viruses. Their location and their differential responses to RNA viruses emphasises the complexity of the innate detection system. Despite the wealth of information on the types of RNA that trigger RIG-I, much less is known about the nature of the RNAs that act as agonists for MDA5. In order to identify which RNA species triggers MDA5 activation during infection, we isolated viral ssRNA and replicative intermediates of RNA from positive sense ssRNA viruses. We reveal that MDA5 recognises not the genomic ssRNA but the dsRNA generated by the replication of these viruses. Furthermore, using fluorescent imaging we present the first report of the visualisation of dsRNA and MDA5, which provides unique evidence of the relationship between viral dsRNA and MDA5 and proves without a doubt that MDA5 is the key sensor for the dsRNA replicative intermediate form of positive sense ssRNA viruses.

Rhinovirus-induced calcium flux triggers NLRP3 and NLRC5 activation in bronchial cells.

Human rhinoviruses have been linked with underlying lung disorders, such as asthma and chronic obstructive pulmonary disease, in children and adults. However, the mechanism of virus-induced airway inflammation is poorly understood. In this study, using virus deletion mutants and silencing for nucleotide-binding oligomerization domain-like receptors (NLRs), we show that the rhinovirus ion channel protein 2B triggers NLRP3 and NLRC5 inflammasome activation and IL-1ß secretion in bronchial cells. 2B protein targets the endoplasmic reticulum and Golgi and induces Ca(2+) reduction in these organelles, thereby disturbing the intracellular calcium homeostasis. NLRP3 and NLRC5 act in a cooperative manner during the inflammasome assembly by sensing intracellular Ca(2+) fluxes and trigger IL-1ß secretion. These results reveal for the first time that human rhinovirus infection in primary bronchial cells triggers inflammasome activation.

Human respiratory syncytial virus viroporin SH: a viral recognition pathway used by the host to signal inflammasome activation.

BACKGROUND: Respiratory syncytial virus (RSV) remains the leading cause of serious viral bronchiolitis and pneumonia in infants and young children throughout the world. The burden of disease is significant, with 70% of all infants being infected with RSV within the first year of their life. 40% of those children discharged from hospital have recurrent, repeated respiratory symptoms and wheezing for at least 10 years. The infection is also an important illness in the elderly and immunocompromised individuals. Ongoing symptoms relate to continued lung inflammation. One cytokine that is associated with RSV infection is IL-1ß, but the mechanism of activation remain unclear. OBJECTIVES: In the current study, we set out to decipher the molecular mechanisms of RSV-induced inflammasome activation. METHODS AND RESULTS: Using deletion mutants of the virus and measuring IL-1ß secretion, as well as caspase 1 expression via western blotting, we demonstrate that the NLRP3 inflammasome is activated through the small hydrophobic (SH) RSV viroporin which induces membrane permeability to ions or small molecules. Confocal microscopy revealed that during virus infection, SH seems to accumulate within lipid rafts in the Golgi compartments. CONCLUSIONS: Upon RSV infection, SH gets localised in the cell membranes and intracellular organelle membranes, and then induces permeability by disrupting membrane architecture, thus leading us to believe that formation of viral ion channels in lipid bilayers of cells is a viral recognition pathway used by the host to signal inflammasome activation.

Divergent age-related humoral correlates of protection against respiratory syncytial virus infection in older and young adults: a pilot, controlled, human infection challenge model.

BACKGROUND: Respiratory viral infections are typically more severe in older adults. Older adults are more vulnerable to infection and do not respond effectively to vaccines due to a combination of immunosenescence, so-called inflamm-ageing, and accumulation of comorbidities. Although age-related changes in immune responses have been described, the causes of this enhanced respiratory disease in older adults remain poorly understood. We therefore performed volunteer challenge with respiratory syncytial virus (RSV) in groups of younger and older adult volunteers. The aim of this study was to establish the safety and tolerability of this model and define age-related clinical, virological, and immunological outcomes. METHODS: In this human infection challenge pilot study, adults aged 18-55 years and 60-75 years were assessed for enrolment using protocol-defined inclusion and exclusion criteria. Symptoms were documented by self-completed diaries and viral load determined by quantitative PCR of nasal lavage. Peripheral blood B cell frequencies were measured by enzyme-linked immunospot and antibodies against pre-fusion and post-fusion, NP, and G proteins in the blood and upper respiratory tract were measured. The study was registered with ClinicalTrials.gov, NCT03728413. FINDINGS: 381 adults aged 60-75 years (older cohort) and 19 adults aged 18-55 years (young cohort) were assessed for enrolment using protocol-defined inclusion and exclusion criteria between Nov 12, 2018, and Feb 26, 2020. 12 healthy volunteers aged 60-75 years and 21 aged 18-55 years were inoculated intranasally with RSV Memphis-37. Nine (67%) of the 12 older volunteers became infected, developing mild-to-moderate upper respiratory tract symptoms that resolved without serious adverse events or sequelae. Viral load peaked on day 6 post-inoculation and symptoms peaked between days 6 and 8. Increases in circulating IgG-positive and IgA-positive antigen-specific plasmablasts, serum neutralising antibodies, and pre-F specific IgG were similar younger and older adults. However, in contrast to young participants, secretory IgA titres in older volunteers failed to increase during infection and, unlike serum IgG, did not correlate with protection. INTERPRETATION: Better understanding of age-related differences in clinical outcomes and immune correlates of protection can overcome reduction in vaccine efficacy with advancing age. We identify correlates of protection in older adults, revealing previously unrecognised factors which might have implications for targeted vaccine discovery and drug development in this vulnerable group. FUNDING: Medical Research Council and GlaxoSmithKline EMINENT Consortium.

Epitope-specific airway-resident CD4+ T cell dynamics during experimental human RSV infection.

BACKGROUNDRespiratory syncytial virus (RSV) is an important cause of acute pulmonary disease and one of the last remaining major infections of childhood for which there is no vaccine. CD4+ T cells play a key role in antiviral immunity, but they have been little studied in the human lung.METHODSHealthy adult volunteers were inoculated i.n. with RSV A Memphis 37. CD4+ T cells in blood and the lower airway were analyzed by flow cytometry and immunohistochemistry. Bronchial soluble mediators were measured using quantitative PCR and MesoScale Discovery. Epitope mapping was performed by IFN-γ ELISpot screening, confirmed by in vitro MHC binding.RESULTSActivated CD4+ T cell frequencies in bronchoalveolar lavage correlated strongly with local C-X-C motif chemokine 10 levels. Thirty-nine epitopes were identified, predominantly toward the 3' end of the viral genome. Five novel MHC II tetramers were made using an immunodominant EFYQSTCSAVSKGYL (F-EFY) epitope restricted to HLA-DR4, -DR9, and -DR11 (combined allelic frequency: 15% in Europeans) and G-DDF restricted to HLA-DPA1*01:03/DPB1*02:01 and -DPA1*01:03/DPB1*04:01 (allelic frequency: 55%). Tetramer labeling revealed enrichment of resident memory CD4+ T (Trm) cells in the lower airway; these Trm cells displayed progressive differentiation, downregulation of costimulatory molecules, and elevated CXCR3 expression as infection evolved.CONCLUSIONSHuman infection challenge provides a unique opportunity to study the breadth of specificity and dynamics of RSV-specific T-cell responses in the target organ, allowing the precise investigation of Trm recognizing novel viral antigens over time. The new tools that we describe enable precise tracking of RSV-specific CD4+ cells, potentially accelerating the development of effective vaccines.TRIAL REGISTRATIONClinicalTrials.gov NCT02755948.FUNDINGMedical Research Council, Wellcome Trust, National Institute for Health Research.