Phenotypic and Functional Characteristics of a Novel Influenza Virus Hemagglutinin-Specific Memory NK Cell.

Immune memory represents the most efficient defense against invasion and transmission of infectious pathogens. In contrast to memory T and B cells, the roles of innate immunity in recall responses remain inconclusive. In this study, we identified a novel mouse spleen NK cell subset expressing NKp46 and NKG2A induced by intranasal influenza virus infection. These memory NK cells specifically recognize N-linked glycosylation sites on influenza hemagglutinin (HA) protein. Different from memory-like NK cells reported previously, these NKp46+ NKG2A+ memory NK cells exhibited HA-specific silence of cytotoxicity but increase of gamma interferon (IFN-γ) response against influenza virus-infected cells, which could be reversed by pifithrin-µ, a p53-heat shock protein 70 (HSP70) signaling inhibitor. During recall responses, splenic NKp46+ NKG2A+ NK cells were recruited to infected lung and modulated viral clearance of virus and CD8+ T cell distribution, resulting in improved clinical outcomes. This long-lived NK memory bridges innate and adaptive immune memory response and promotes the homeostasis of local environment during recall response.IMPORTANCE In this study, we demonstrate a novel hemagglutinin (HA)-specific NKp46+ NKG2A+ NK cell subset induced by influenza A virus infection. These memory NK cells show virus-specific decreased cytotoxicity and increased gamma interferon (IFN-γ) on reencountering the same influenza virus antigen. In addition, they modulate host recall responses and CD8 T cell distribution, thus bridging the innate immune and adaptive immune responses during influenza virus infection.

CLEC5A-Mediated Enhancement of the Inflammatory Response in Myeloid Cells Contributes to Influenza Virus Pathogenicity In Vivo.

Human infections with influenza viruses exhibit mild to severe clinical outcomes as a result of complex virus-host interactions. Induction of inflammatory mediators via pattern recognition receptors may dictate subsequent host responses for pathogen clearance and tissue damage. We identified that human C-type lectin domain family 5 member A (CLEC5A) interacts with the hemagglutinin protein of influenza viruses expressed on lentiviral pseudoparticles through lectin screening. Silencing CLEC5A gene expression, blocking influenza-CLEC5A interactions with anti-CLEC5A antibodies, or dampening CLEC5A-mediated signaling using a spleen tyrosine kinase inhibitor consistently reduced the levels of proinflammatory cytokines produced by human macrophages without affecting the replication of influenza A viruses of different subtypes. Infection of bone marrow-derived macrophages from CLEC5A-deficient mice showed reduced levels of tumor necrosis factor alpha (TNF-α) and IP-10 but elevated alpha interferon (IFN-α) compared to those of wild-type mice. The heightened type I IFN response in the macrophages of CLEC5A-deficient mice was associated with upregulated TLR3 mRNA after treatment with double-stranded RNA. Upon lethal challenges with a recombinant H5N1 virus, CLEC5A-deficient mice showed reduced levels of proinflammatory cytokines, decreased immune cell infiltration in the lungs, and improved survival compared to the wild-type mice, despite comparable viral loads noted throughout the course of infection. The survival difference was more prominent at a lower dose of inoculum. Our results suggest that CLEC5A-mediated enhancement of the inflammatory response in myeloid cells contributes to influenza pathogenicity in vivo and may be considered a therapeutic target in combination with effective antivirals. Well-orchestrated host responses together with effective viral clearance are critical for optimal clinical outcome after influenza infections. IMPORTANCE: Multiple pattern recognition receptors work in synergy to sense viral RNA or proteins synthesized during influenza replication and mediate host responses for viral control. Well-orchestrated host responses may help to maintain the inflammatory response to minimize tissue damage while inducing an effective adaptive immune response for viral clearance. We identified that CLEC5A, a C-type lectin receptor which has previously been reported to mediate flavivirus-induced inflammatory responses, enhanced induction of proinflammatory cytokines and chemokines in myeloid cells after influenza infections. CLEC5A-deficient mice infected with influenza virus showed reduced inflammation in the lungs and improved survival compared to that of the wild-type mice despite comparable viral loads. The survival difference was more prominent at a lower dose of inoculum. Collectively, our results suggest that dampening CLEC5A-mediated inflammatory responses in myeloid cells reduces immunopathogenesis after influenza infections.

The R292K mutation that confers resistance to neuraminidase inhibitors leads to competitive fitness loss of A/Shanghai/1/2013 (H7N9) influenza virus in ferrets.

BACKGROUND: Neuraminidase (NA) inhibitors are the only licensed therapeutic option for human zoonotic H7N9 infections. An NA-R292K mutation that confers broad-spectrum resistance to NA inhibitors has been documented in H7N9 patients after treatment. METHODS: We evaluated the transmission potential of a human influenza A H7N9 isolate with a NA-R292K mutation in the ferret model followed by genotyping assay to monitor its competitive fitness in vivo. RESULTS: Plaque-purified A/Shanghai/1/2013 wild-type and NA-R292K viruses transmitted at comparable efficiency to direct or respiratory droplet contact ferrets. In ferrets inoculated with the plaque-purified A/Shanghai/1/2013 NA-R292K virus with dominant K292 (94%), the resistant K292 genotype was outgrown by the wild-type R292 genotype during the course of infection. Transmission of the resistant K292 genotype was detected in 3/4 direct contact and 3/4 respiratory droplet contact ferrets at early time points but was gradually replaced by the wild-type genotype. In the respiratory tissues of inoculated or infected ferrets, the wild-type R292 genotype dominated in the nasal turbinate, whereas the resistant K292 genotype was more frequently detected in the lungs. CONCLUSIONS: The NA inhibitor-resistant H7N9 virus with the NA-R292K mutation may transmit among ferrets but showed compromised fitness in vivo while in competition with the wild-type virus.

Biomarkers of early SARS-CoV-2 infection before the onset of respiratory symptoms.

OBJECTIVES: Currently, limited data exist regarding the pathological changes occurring during the incubation phase of SARS-CoV-2 infection. We utilized proteomic analysis to explore changes in the circulatory host response in individuals with SARS-CoV-2 infection before the onset of symptoms. METHODS: Participants were individuals from a randomized clinical trial of prophylaxis for COVID-19 in a workers' dormitory. Proteomic signatures of blood samples collected within 7 days before symptom onset (incubation group) were compared with those collected >21 days (non-incubation group) to derive candidate biomarkers of incubation. Candidate biomarkers were assessed by comparing levels in the incubation group with both infected individuals (positive controls) and non-infected individuals (negative controls). RESULTS: The study included men (mean age 34.2 years and standard deviation 7.1) who were divided into three groups: an incubation group consisting of 44 men, and two control groups-positive (n = 56) and negative (n = 67) controls. Through proteomic analysis, we identified 49 proteins that, upon pathway analyses, indicated an upregulation of the renin-angiotensin and innate immune systems during the virus incubation period. Biomarker analyses revealed increased concentrations of plasma angiotensin II (mean 731 vs. 139 pg/mL), angiotensin (1-7) (302 vs. 9 pg/mL), CXCL10 (423 vs. 85 pg/mL), CXCL11 (82.7 vs. 32.1 pg/mL), interferon-gamma (0.49 vs. 0.20 pg/mL), legumain (914 vs. 743 pg/mL), galectin-9 (1443 vs. 836 pg/mL), and tumour necrosis factor (20.3 vs. 17.0 pg/mL) during virus incubation compared with non-infected controls (all p < 0.05). Plasma angiotensin (1-7) exhibited a significant increase before the onset of symptoms when compared with uninfected controls (area under the curve 0.99, sensitivity 0.97, and specificity 0.99). DISCUSSION: Angiotensin (1-7) could play a crucial role in the progression of symptomatic COVID-19 infection, and its assessment could help identify individuals who would benefit from enhanced monitoring and early antiviral intervention.

Plasma NOTCH3 and the risk of cardiovascular recurrence in patients with ischemic stroke.

BACKGROUND: Ischemic stroke patients are more prone to developing another cardiovascular event. AIM: This study aims to examine potential biological predispositions to cardiovascular recurrence in patients with ischemic stroke. DESIGN: Human and preclinical studies. METHODS: Quantitative proteomic analysis, animal stroke, atherosclerosis models and circulating endothelial cells (CECs) were employed to examine candidate biomarkers derived from an ischemic stroke cohort in Singapore. RESULTS: Proteomic analysis of pooled microvesicles of "Event" (n = 24) and without "Event" (n = 24) samples identified NOTCH3 as a candidate marker; plasma NOTCH3 were shown to be elevated in "Event" patients compared to those without "Events" and age-matched controls. In a validation cohort comprising 431 prospectively recruited ischemic stroke patients (mean age 59.1 years; median follow-up 3.5 years), men with plasma NOTCH3 (>1600pg/ml) harbored increased risk of cardiovascular recurrence (adjusted hazards ratio 2.29, 95% CI 1.10-4.77); no significant association was observed in women. Chronic renal failure, peripheral artery disease and NT-pro-brain natriuretic peptide were significant predictors of plasma NOTCH3 in men without ischemic stroke (adjusted r2=0.43). Following middle cerebral artery occlusion, NOTCH3 expression in mouse sera increased and peaked at 24 hrs, persisting thereafter for at least 72 hours. In Apoe-/- atherosclerotic mice, NOTCH3 stained the endothelium of defective arterial lining and atherosclerotic plaques. Analysis of CECs isolated from stroke patients revealed increased gene expression of NOTCH3, further supporting endothelial damage underpinning NOTCH3-mediated atherosclerosis. CONCLUSION: Findings from this study suggests that NOTCH3 could be important in cardiovascular recurrence following an ischemic stroke.

Hybrid immunity augments cross-variant protection against COVID-19 among immunocompromised individuals.

BACKGROUND: Immunity to SARS-CoV-2 vaccination and infection differs considerably among individuals. We investigate the critical pathways that influence vaccine-induced cross-variant serological immunity among individuals at high-risk of COVID-19 complications. METHODS: Neutralizing antibodies to the wild-type SARS-CoV-2 virus and its variants (Beta, Gamma, Delta and Omicron) were analyzed in patients with autoimmune diseases, chronic comorbidities (multimorbidity), and healthy controls. Antibody levels were assessed at baseline and at different intervals up to 12 months following primary and booster vaccination with either BNT162b2 or mRNA-1273. Immunity induced by vaccination with and without infection (hybrid immunity) was compared with that of unvaccinated individuals with recent SARS-CoV-2 infection. Plasma cytokines were analyzed to investigate variations in antibody production following vaccination. RESULTS: Patients with autoimmune diseases (n = 137) produced lesser antibodies to the wild-type SARS-CoV-2 virus and its variants compared with those in the multimorbidity (n = 153) and healthy groups (n = 229); antibody levels were significantly lower in patients with neuromyelitis optica and those on prednisolone, mycophenolate or rituximab treatment. Multivariate logistic regression analysis identified neuromyelitis optica (odds ratio 8.20, 95% CI 1.68-39.9) and mycophenolate (13.69, 3.78-49.5) as significant predictors of a poorer antibody response to vaccination (i.e, neutralizing antibody <40%). Infected participants exhibited antibody levels that were 28.7% higher (95% CI 24.7-32.7) compared to non-infected participants six months after receiving a booster vaccination. Individuals infected during the Delta outbreak generated cross-protective neutralizing antibodies against the Omicron variant in quantities comparable to those observed after infection with the Omicron variant itself. In contrast, unvaccinated individuals recently infected with the wild-type (n = 2390) consistently displayed lower levels of neutralizing antibodies against both the wild-type virus and other variants. Pathway analyses suggested an inverse relationship between baseline T cell subsets and antibody production following vaccination. CONCLUSION: Hybrid immunity confers a robust protection against COVID-19 among immunocompromised individuals.

Repurposing povidone-iodine to reduce the risk of SARS-CoV-2 infection and transmission: a narrative review.

BACKGROUND: Accumulating data suggest antiviral effects of povidone-iodine against the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. This narrative review aims to examine the antiviral mechanisms of povidone-iodine, efficacy of povidone-iodine against the SARS-CoV-2 virus, and safety of povidone-iodine to human epithelial cells and thyroid function. METHODS: We searched the electronic databases PubMed, Embase, Cochrane Library, ClinicalTrials.gov and World Health Organization's International Clinical Trials Registry Platform for articles containing the keywords "povidone-iodine", "SARS-CoV-2" and "COVID-19" from database inception till 3 June 2021. RESULTS: Despite in vitro data supporting the anti-SARS-CoV-2 effects of povidone-iodine, findings from clinical studies revealed differences in treatment response depending on study settings (healthy vs. hospitalized individuals), treatment target (nasal vs. oral vs. pharynx), method of administration (oral rinse vs. gargle vs. throat spray) and choice of samples used to measure study endpoints (nasopharyngeal vs. saliva). One large-scale clinical trial demonstrated reduction in the incidence of SARS-CoV-2 infection among participants who administered povidone-iodine 3 times daily during an active outbreak. Povidone-iodine is also used to disinfect the oro-pharyngeal space prior to dental or otolaryngology procedures. Although existing data suggest minimal impact of povidone-iodine on thyroid function, high-quality safety data are presently lacking. CONCLUSIONS: Povidone-iodine application to the oropharyngeal space could complement existing non-pharmacological interventions to reduce SARS-CoV-2 infection especially in high exposure settings.Key messagesAccumulating data suggest antiviral effects of povidone-iodine against the SARS-CoV-2 virus.Findings from clinical studies reveal differences in treatment response depending on study settings, treatment target, method of administration and choice of samples used to measure study endpoints. One large-scale clinical trial observed reduction in the incidence of SARS-CoV-2 infection among participants who administered povidone-iodine 3 times daily during an active outbreak.Povidone-iodine application to the oropharyngeal space could complement existing non-pharmacological interventions to reduce SARS-CoV-2 infection especially in high exposure settings.

Stable thyroid function despite regular use of povidone-iodine throat spray for SARS-CoV-2 prophylaxis.

BACKGROUND: It is unclear whether unintentional ingestion of povidone-iodine following its application to the oropharyngeal space could affect thyroid function. OBJECTIVE: To examine thyroid function among individuals who regularly apply povidone-iodine throat spray for SARS-CoV-2 prophylaxis. METHODS: We designed a case-control study to compare thyroid function among participants who received povidone-iodine throat spray three times a day for 42 days ('cases') and those who received vitamin C ('controls'). Thyroid function was assessed by profiling serum TSH, free T3, and free T4; iodine status was estimated using serum thyroglobulin level, while infection status was determined by measuring anti-SARS-CoV-2 antibody against the nucleocapsid antigen. All measurements were performed in pairs, at baseline and 42 days later. Pre-post changes in thyroid function were compared between groups, before and after stratification according to baseline TSH quartiles. RESULTS: A total of 177 men (117 cases and 60 controls) (mean age, 32.2 years) were included. Despite comparable demographics and clinical profiles, no clinically or statistically significant differences were observed in thyroid indices between 'cases' and 'controls' before and after stratification according to TSH quartiles. None of the participants developed symptomatic hypo- or hyperthyroidism throughout the study. Post-hoc analysis did not reveal differences in thyroid function according to infection status. CONCLUSIONS: Data from this study support the overall safety of povidone-iodine use in the oropharyngeal space for SARS-CoV-2 prophylaxis among individuals with normal thyroid function and subclinical thyroid disease.

Macrophage-Bacteria Interactions-A Lipid-Centric Relationship.

Macrophages are professional phagocytes at the front line of immune defenses against foreign bodies and microbial pathogens. Various bacteria, which are responsible for deadly diseases including tuberculosis and salmonellosis, are capable of hijacking this important immune cell type and thrive intracellularly, either in the cytoplasm or in specialized vacuoles. Tight regulation of cellular metabolism is critical in shaping the macrophage polarization states and immune functions. Lipids, besides being the bulk component of biological membranes, serve as energy sources as well as signaling molecules during infection and inflammation. With the advent of systems-scale analyses of genes, transcripts, proteins, and metabolites, in combination with classical biology, it is increasingly evident that macrophages undergo extensive lipid remodeling during activation and infection. Each bacterium species has evolved its own tactics to manipulate host metabolism toward its own advantage. Furthermore, modulation of host lipid metabolism affects disease susceptibility and outcome of infections, highlighting the critical roles of lipids in infectious diseases. Here, we will review the emerging roles of lipids in the complex host-pathogen relationship and discuss recent methodologies employed to probe these versatile metabolites during the infection process. An improved understanding of the lipid-centric nature of infections can lead to the identification of the Achilles' heel of the pathogens and host-directed targets for therapeutic interventions. Currently, lipid-moderating drugs are clinically available for a range of non-communicable diseases, which we anticipate can potentially be tapped into for various infections.