Acute Influenza Virus Infection Research Articles

FcγRIIB modulates splenic germinal center response against immune subversion during acute influenza A virus infection

BackgroundB cells are essential for providing humoral protection against acute influenza A virus (IAV) infection. FcγRIIB, a regulator of antibody (Ab) production, influences immune responses during pathogen infections, but its specific impact on humoral protection and B cell-mediated responses against IAV remains unclear. MethodsTo investigate FcγRIIB's role in host defense and B cell function during acute IAV infection, we generated mice with systemic FcγRIIB deficiency, functional impairment, and B cell-specific FcγRIIB deletion. We infected these mice with PR8 (H1N1) or Hkx31 (H3N2) IAVs and evaluated body weight preservation, survival rates, Ab production, viral neutralization, Ab affinity maturation, and germinal center B cell development. ResultsMice lacking FcγRIIB or with impaired function showed improved protection, preserved body weight, and increased survival rates during IAV infection. Notably, mice with haploinsufficient FcγRIIB function displayed protective effects. Selective deficiency of FcγRIIB in B cells led to enhanced Ab production, resulting in elevated IAV-specific Abs in the serum with superior viral neutralizing potency. However, the impact on the affinity maturation index of virus-specific Abs was modest. Accordingly, FcγRIIB-deficient B cells maintained normal germinal center B cell development during IAV infection, whereas wild-type mice exhibited delayed differentiation. ConclusionOur research underscores the pivotal role of FcγRIIB in host defense and B cell-mediated immunity during acute IAV infection. Additionally, our discoveries hold implications for antiviral treatments, particularly during the initial stages of IAV infection, aimed at enhancing the host's humoral immune response.

Determination of Tr1 cell populations correlating with distinct activation states in acute IAV infection

Type I regulatory (Tr1) cells are defined as FOXP3-IL-10-secreting CD4+ T cells that contribute to immune suppression and typically express the markers LAG-3 and CD49b and other co-inhibitory receptors. These cells have not been studied in detail the context of the resolution of acute infection in the lung. Here, we identify FOXP3-IL-10+ CD4+ T cells transiently accumulating in the lung parenchyma during resolution of the response to sublethal influenza A virus (IAV) infection in mice. These cells were dependent on IL-27Rα, which was required for timely recovery from IAV-induced weight loss. LAG-3 and CD49b were not generally co-expressed by FOXP3- IL-10+ CD4+ T cells in this model and four populations of these cells based on LAG-3 and CD49b co-expression were apparent (LAG-3-CD49b- [DN], LAG-3+CD49b+ [DP], LAG-3+CD49b- [LAG-3+], LAG-3-CD49b+ [CD49b+]). However, each population exhibited suppressive potential consistent with the definition of Tr1 cells. Notably, differences between these populations of Tr1 cells were apparent including differential dependence on IL-10 to mediate suppression and expression of markers indicative of different activation states and terminal differentiation. Sort-transfer experiments indicated that LAG-3+ Tr1 cells exhibited the capacity to convert to DN and DP Tr1 cells, indicative of plasticity between these populations. Together, these data determine the features and suppressive potential of Tr1 cells in the resolution of IAV infection and identify four populations delineated by LAG-3 and CD49b, which likely correspond to different Tr1 cell activation states.

2189. Anti-inflammatory Activity of Lefamulin in a Mouse Model of Influenza Virus H1N1 Infection

Abstract Background Lefamulin (LEF) is a pleuromutilin antibiotic approved by the United States (US) FDA for the treatment of community-acquired bacterial pneumonia in adults. In addition to the potent antibacterial activity, LEF has demonstrated anti-inflammatory activity in an LPS induced lung neutrophilia model in mice. We investigated the anti-inflammatory activity of lefamulin in the H1N1 influenza mouse model in comparison to oseltamivir (OTV) and azithromycin (AZM). Methods Infection was performed in BALB/c mice by intranasal challenge ∼70 pfu influenza virus H1N1 A/PR/8/1934 (Day 0). Treatment with drugs at clinically relevant doses started on Day -1 (LEF 70 mg/kg/day and 110/140 mg/kg/day, s.c., AZM 30 mg/kg/day, i,p. and OTV 20 mg/kg/day p.o.) to Day 6. On Days 3 and 6, bronchioalveolar lavage fluid (BALF) was collected to measure infiltrating lung leukocytes and cytokines. Lung immunopathology following infection was evaluated on Day 6 by gross pathology at termination together with hematoxylin and eosin histopathology. Results In untreated vehicle control animals, the influenza infection progressed as expected with bodyweight loss, increased cell infiltration into the lung and increased levels of TNF-α, IL-6 at day 3 and 6. Treatment with LEF significantly decreased the total immune cell infiltration into the lung by day 6 at both doses tested (Figure Left). Cytokine levels in the BALF were significantly reduced on day 3 when the viral load peaked. Furthermore, LEF showed positive effects on lung gross pathology and survival. Oseltamivir and LEF, at both doses, appeared efficacious in the suppression of the development of influenza-induced bronchi-interstitial pneumonia, whereas azithromycin didn’t show reduced pathology (Figure Right). Anti-inflammatory effects of LEF, AZM and OTV in BALF of H1N1 infected mice infected with influenza virus H1N1. Left: Total lung leukocyte infiltrate following infection on day 0. Bars represent mean ± SEM. Right: Day 6 lung inflammation and degeneration histopathology score. Data are presented as mean histopathology score + SEM (n=10). One-way ANOVA, with Dunnett’s multiple comparisons against the vehicle control treatment was run for each timepoint. * represents significance level of p<0.05. ** indicates p<0.01. Conclusion Lefamulin showed anti-inflammatory treatment following acute influenza virus infection. Following influenza infection LEF was able to significantly reduce lung immunopathology and improve clinical outcome in mice. Results from this experiment were consistent with that observed in the LPS induced lung neutrophilia mouse model. Further studies are warranted to evaluate the immunomodulatory potential of Lefamulin. Disclosures Wolfgang W. Wicha, MSc, Nabriva Therapeutics: Grant/Research Support|Nabriva Therapeutics: Inventor|Nabriva Therapeutics: Employee|Nabriva Therapeutics: Stocks/Bonds Sandy Kimber, PhD, Nabriva Therapeutics: Grant/Research Support Charlotte Cumper, BSc, Nabriva Therapeutics: Grant/Research Support Hon S. Lam, MSc, Nabriva Therapeutics: Grant/Research Support Lorena S. Ballesteros, MSc, Nabriva Therapeutics: Grant/Research Support Christopher Kirkham, PhD, Nabriva Therapeutics: Grant/Research Support Claire Richards, PhD, Nabriva Therapeutics: Grant/Research Support Steven P. Gelone, PharmD, Nabriva Therapeutics: Board Member|Nabriva Therapeutics: Inventor|Nabriva Therapeutics: Employee|Nabriva Therapeutics: Stocks/Bonds|Nabriva Therapeutics: Stocks/Bonds Susanne Paukner, PhD, Nabriva Therapeutics: Inventor|Nabriva Therapeutics: Employee|Nabriva Therapeutics: Stocks/Bonds.

A multicenter cohort study of severe dengue and critically ill influenza patients with elevated cardiac troponin-I: Difference clinical features and high mortality

BackgroundAs cardiac involvement can cause serious complications and death, understanding its role in acute dengue and influenza virus infections is important. MethodsWe provide a comparative evaluation of severe dengue and critically ill influenza patients with elevated cardiac troponin-I (cTnI) from 2014 to 2019. Inclusion criteria included patients in which cTnI test were ordered. Patient without cTnI test was excluded. ResultsDuring the study period, 82 (41 severe dengue and 41 critically ill influenza) patients had cTnI elevations, and 81 (35 severe dengue and 46 critically ill influenza) patients had a single normal cTnI test. Severe dengue patients with cTnI elevations had a significantly higher incidences of acute kidney injury, gastrointestinal bleeding, early mortality (≤7 after illness onset) and in-hospital mortality than those with severe dengue and single normal cTnI test. Significantly higher aspartate aminotransferase (AST) levels and higher incidence of gastrointestinal bleeding was observed in critically ill influenza patients with cTnI elevations compared to critically ill influenza patients with single normal cTnI measurement. Of the patients with cTnI elevations, the early and in-hospital mortality rates were 53.6% and 65.8%, respectively, in severe dengue patients, and 7.3% and 46.3%, respectively, in critically ill influenza patients. Significantly higher early mortality rates were observed in severe dengue patients with elevated cTnI levels than in critically ill influenza patients with cTnI elevations. Critically ill influenza patients with elevated cTnI levels had significantly higher incidences of pneumonia, pneumothorax, and bacteremia than severe dengue patients with cTnI elevations. Multivariate analysis revealed elevated AST (>1000U/L) (95% confidence interval [CI]: 1.690–143.174) was an independent risk factor for in-hospital mortality in severe dengue patients with elevated cTnI levels. Leukocytosis (95% CI: 1.079–1.124) and thrombocytopenia (95% CI: 2.739–5.821) were independently correlated with in-hospital mortality in critically ill influenza patients with cTnI elevations. ConclusionsDifferences in clinical features between severe dengue and critically ill influenza patients with cTnI elevations. High early mortality rate was observed in severe dengue patients with cardiac involvement. In contrast, most critically ill influenza patients died ≥2 weeks after the onset of illness, regardless of cTnI elevations. Our report has important clinical implications for the timely recognition and management of cardiac complication in patients with acute dengue and influenza virus infections.

Natural killer cells in antiviral immunity.

Natural killer (NK) cells play an important role in innate immune responses to viral infections. Here, we review recent insights into the role of NK cells in viral infections, with particular emphasis on human studies. We first discuss NK cells in the context of acute viral infections, with flavivirus and influenza virus infections as examples. Questions related to activation of NK cells, homing to infected tissues and the role of tissue-resident NK cells in acute viral infections are also addressed. Next, we discuss NK cells in the context of chronic viral infections with hepatitis C virus and HIV-1. Also covered is the role of adaptive-like NK cell expansions as well as the appearance of CD56− NK cells in the course of chronic infection. Specific emphasis is then placed in viral infections in patients with primary immunodeficiencies affecting NK cells. Not least, studies in this area have revealed an important role for NK cells in controlling several herpesvirus infections. Finally, we address new data with respect to the activation of NK cells and NK cell function in humans infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) giving rise to coronavirus disease 2019 (COVID-19).

Regulatory KIR+ RA+ T cells accumulate with age and are highly activated during viral respiratory disease.

Severe respiratory viral infectious diseases such as influenza and COVID‐19 especially affect the older population. This is partly ascribed to diminished CD8+ T‐cell responses a result of aging. The phenotypical diversity of the CD8+ T‐cell population has made it difficult to identify the impact of aging on CD8+ T‐cell subsets associated with diminished CD8+ T‐cell responses. Here we identify a novel human CD8+ T‐cell subset characterized by expression of Killer‐cell Immunoglobulin‐like Receptors (KIR+) and CD45RA (RA+). These KIR+RA+ T cells accumulated with age in the blood of healthy individuals (20–82 years of age, n = 50), expressed high levels of aging‐related markers of T‐cell regulation, and were functionally capable of suppressing proliferation of other CD8+ T cells. Moreover, KIR+RA+ T cells were a major T‐cell subset becoming activated in older adults suffering from an acute respiratory viral infection (n = 36), including coronavirus and influenza virus infection. In addition, older adults with influenza A infection showed that higher activation status of their KIR+RA+ T cells associated with longer duration of respiratory symptoms. Together, our data indicate that KIR+RA+ T cells are a unique human T‐cell subset with regulatory properties that may explain susceptibility to viral respiratory disease at old age.

Latent CMV Infection Is Associated With Lower Influenza Virus-Specific Memory T-Cell Frequencies, but Not With an Impaired T-Cell Response to Acute Influenza Virus Infection.

Latent infection with cytomegalovirus (CMV) is assumed to contribute to the age-associated decline of the immune system. CMV induces large changes in the T-cell pool and may thereby affect other immune responses. CMV is expected to impact especially older adults, who are already at higher risk of severe disease and hospitalization upon infections such as influenza virus (IAV) infection. Here, we investigated the impact of CMV infection on IAV-specific CD8+ T-cell frequencies in healthy individuals (n=96) and the response to IAV infection in older adults (n=72). IAV-specific memory T-cell frequencies were lower in healthy CMV+ older individuals compared to healthy CMV- older individuals. Upon acute IAV infection, CMV serostatus or CMV-specific antibody levels were not negatively associated with IAV-specific T-cell frequencies, function, phenotype or T-cell receptor repertoire diversity. This suggests that specific T-cell responses upon acute IAV infection are not negatively affected by CMV. In addition, we found neither an association between CMV infection and inflammatory cytokine levels in serum during acute IAV infection nor between cytokine levels and the height of the IAV-specific T-cell response upon infection. Finally, CMV infection was not associated with increased severity of influenza-related symptoms. In fact, CMV infection was even associated with increased IAV-specific T-cell responses early upon acute IAV infection. In conclusion, although associated with lower frequencies of memory IAV-specific T cells in healthy individuals, CMV infection does not seem to hamper the induction of a proper T-cell response during acute IAV infection in older adults.

Role of IL-17-EGFR axis in alleviated lung inflammation and recovery during the late phase of acute influenza virus infection

Abstract Influenza hemagglutinin (HA)-specific T-Cell-Receptor (TCR) transgenic mice sustain the influenza virus infection of an inoculum size which is fatal in wild type mice. There is a prominent IL-17 response of the HA-specific TCR transgenic CD4+ T cells in late phase of infection in the transgenic mice. We have demonstrated that the late phase IL-17 response is a de novo response of naïve T cells under guidance of the Th1 cells with influenza viral neuraminidase-activated TGF-β. There is also a late phase up-regulation of epidermal growth factor receptor (EGFR) on the transgenic CD4+ T cells in the lungs. EGFR inhibition with Gefitinib resulted in impaired sustainability of the infection of the transgenic mice. They suffered from profound body weight loss and severe disease with significant mortality. IL-17-induced EGFR-mediated signaling cascade has been documented in skin stem cells that are involved in wound healing. We will further dissect the role of this cascade in alleviation of lung inflammation and recovery from the disease during the late phase of acute influenza virus infection.

Host DNA released by NETosis in neutrophils exposed to seasonal H1N1 and highly pathogenic H5N1 influenza viruses

BackgroundNeutrophil is of the most abundant number in human immune system. During acute influenza virus infection, neutrophils are already active in the early phase of inflammation - a time in which clinical biopsy or autopsy material is not readily available. However, the role of neutrophil in virus infection is not well understood. Here, we studied the role of neutrophil in host defense during influenza A virus infection, specifically assessing if it contributes to the differential pathogenesis in H5N1 disease.MethodsNeutrophils were freshly isolated from healthy volunteers and subjected to direct influenza H1N1 and H5N1 virus infection in vitro. The ability of the naïve neutrophils to infiltrate from the basolateral to the apical phase of the influenza virus infected alveolar epithelium was assessed. The viral replication, innate immune responses and Neutrophil extracellular trap (NET) formation of neutrophils upon influenza virus infection were evaluated.ResultsOur results demonstrated that influenza virus infected alveolar epithelium allowed neutrophil transmigration. Significantly more neutrophils migrated across the H5N1 influenza virus infected the epithelium than the counterpart infected by the seasonal influenza H1N1 virus infected. Neutrophils were equally susceptible to H5N1 and H1N1 virus infection with similar viral gene transcription. Productive replication was observed in H5N1 infected neutrophils. H5N1 induced higher cytokine and chemokine gene transcription than H1N1 infected neutrophils, including TNF-α, IFN-β, CXCL10, MIP-1α and IL-8. This inferred a more intense inflammatory response posed by H5N1 than H1N1 virus. Strikingly, NADPH oxidase-independent NET formation was only observed in H1N1 infected neutrophils at 6 hpi while no NET formation was observed upon H5N1 infection.ConclusionOur data is the first to demonstrate that NET formation is abrogated in H5N1 influenza virus infection and might contribute to the severity of H5N1 disease.

Efficacy and safety of convalescent plasma for severe COVID-19 based on evidence in other severe respiratory viral infections: a systematic review and meta-analysis.

The safety and efficacy of convalescent plasma in severe coronavirus disease 2019 (COVID-19) remain uncertain. To support a guideline on COVID-19 management, we conducted a systematic review and meta-analysis of convalescent plasma in COVID-19 and other severe respiratory viral infections. In March 2020, we searched international and Chinese biomedical literature databases, clinical trial registries and prepublication sources for randomized controlled trials (RCTs) and nonrandomized studies comparing patients receiving and not receiving convalescent plasma. We included patients with acute coronavirus, influenza and Ebola virus infections. We conducted a meta-analysis using random-effects models and assessed the quality of evidence using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. Of 1099 unique records, 6 studies were eligible, and none of these included patients with COVID-19. One nonrandomized study (n = 40) on convalescent plasma in severe acute respiratory syndrome coronavirus (SARS-CoV) provided uninformative results regarding mortality (relative risk [RR] 0.10, 95% confidence interval [CI] CI 0.01 to 1.70). Pooled estimates from 4 RCTs on influenza (n = 572) showed no convincing effects on deaths (4 RCTs, RR 0.94, 95% CI 0.49 to 1.81), complete recovery (2 RCTs, odds ratio 1.04, 95% CI 0.69 to 1.64) or length of stay (3 RCTs, mean difference -1.62, 95% CI -3.82 to 0.58, d). The quality of evidence was very low for all efficacy outcomes. Convalescent plasma caused few or no serious adverse events in influenza RCTs (RR 0.85, 95% CI 0.56 to 1.29, low-quality evidence). Studies of non-COVID-19 severe respiratory viral infections provide indirect, very low-quality evidence that raises the possibility that convalescent plasma has minimal or no benefit in the treatment of COVID-19 and low-quality evidence that it does not cause serious adverse events.

Combination immunotherapy with anti-PD-L1 antibody and depletion of regulatory T cells during acute viral infections results in improved virus control but lethal immunopathology.

Combination immunotherapy (CIT) is currently applied as a treatment for different cancers and is proposed as a cure strategy for chronic viral infections. Whether such therapies are efficient during an acute infection remains elusive. To address this, inhibitory receptors were blocked and regulatory T cells depleted in acutely Friend retrovirus-infected mice. CIT resulted in a dramatic expansion of cytotoxic CD4+ and CD8+ T cells and a subsequent reduction in viral loads. Despite limited viral replication, mice developed fatal immunopathology after CIT. The pathology was most severe in the gastrointestinal tract and was mediated by granzyme B producing CD4+ and CD8+ T cells. A similar post-CIT pathology during acute Influenza virus infection of mice was observed, which could be prevented by vaccination. Melanoma patients who developed immune-related adverse events under immune checkpoint CIT also presented with expanded granzyme-expressing CD4+ and CD8+ T cell populations. Our data suggest that acute infections may induce immunopathology in patients treated with CIT, and that effective measures for infection prevention should be applied.

Acute Macular Neuroretinopathy Associated with Acute Influenza Virus Infection

ABSTRACT Purpose: To describe a prospective case series of patients with acute macular neuroretinopathy (AMN) associated with acute influenza virus infection Methods: Patients who presented with acute macular neuroretinopathy associated with confirmed influenza virus infection were subject to a detailed clinical history, HLA typing and longitudinal ophthalmological and imaging examinations. Results: Four female patients aged 18 to 32 years were studied. They reported the onset of ocular symptoms between 2 and 5 days after the development of flu like symptoms. Three patients had confirmed acute influenza B infection, while the fourth had influenza A. OCT angiography only demonstrated abnormal choriocapillaris perfusion in 1 patient and early oral Oseltamivir treatment appeared not to affect the ophthalmic outcome in one patient. Conclusion: This is the first report of AMN associated with virologically confirmed acute influenza virus infection. Variation in HLA alleles do not appear to predispose patients to influenza associated AMN.

David Versus Goliath: The Clinical Consequences of an Influenza A Viral Infection in an Unvaccinated Patient

Acute influenza virus (AIV) infection can manifest as a severe life-threating illness in patients who are not vaccinated, and furthermore, have comorbidities that place them at risk for rapid respiratory decompensation. Each year influenza causes death in individuals with high risk for contracting this infection, although the illness is preventable by vaccination. Complications of AIV infection, such as bacterial pneumonia are treatable, but other severe complications such as acute respiratory distress syndrome (ARDS) leading to diffuse alveolar damage (DAD) are limited to supportive therapy and self-resolution. In most cases, ARDS leading to DAD is fatal, due to the insidious severity of symptoms which lead to rapid oxygen desaturation without correction, and despite supportive therapy. Regardless of a poor prognosis, the clinical signs and symptoms are congruent with imaging and attest to the importance of vaccination, which protect against high mortality rates.

Nasal commensal Staphylococcus epidermidis enhances interferon-\u03bb-dependent immunity against influenza virus

BackgroundStaphylococcus epidermidis is one of the most abundant colonizers of healthy human mucosa including that in the respiratory tract. As the respiratory microbiome has been linked to host immune responses, this study sought to determine the role of nasal mucosa-associated S. epidermidis in innate immune responses against the influenza A virus (IAV). S. epidermidis strains were isolated from nasal mucus samples of healthy individuals. The effects of these mucosa-derived commensal strains on interferon (IFN)-dependent innate immunity and IAV infection dynamics were tested in vitro using normal human nasal epithelial (NHNE) cells and human turbinate mucosa. The effects of S. epidermidis on antiviral immunity were also tested in vivo using an acute IAV infection mouse model.ResultsExposure of NHNE cells to nasal mucosa-derived S. epidermidis increased IFN-λ mRNA and secreted protein levels in the absence of viral stimulation. In the context of IAV infection, NHNE exposure to S. epidermidis prevented an increase in the viral burden, as revealed by IAV PA mRNA abundance, IAV nucleoprotein levels, and viral titers. S. epidermidis also enhanced transcription of IFN-stimulated genes independently of Toll-like receptor 2 and further induced IFN-λ production in IAV-infected cells by promoting phosphorylation of interferon regulatory factor 7. In a murine infection model, S. epidermidis prevented the spread of IAV to the lungs by stimulating IFN-λ innate immunity and suppressing IAV replication in the nasal mucosa.ConclusionThe human nasal commensal S. epidermidis mediates front-line antiviral protection against IAV infection through modulation of IFN-λ-dependent innate immune mechanisms in the nasal mucosa, thereby demonstrating the role of host-bacterial commensalism in shaping human antiviral responses.

Influenza A virus (IAV) infection in humans leads to expansion of highly diverse CD8 T cell repertoires cross-reactive with Epstein Barr virus (EBV)

Abstract The competence of T cell responses predominantly depends on how efficient T cell receptors (TCRs) are at recognizing antigenic epitopes. We show here that during acute severely symptomatic IAV infection there was an expansion of IAV-M1/EBV-BRLF1 and IAV-M1/EBV-BMLF1 double-tetramer+ cells directly ex vivo in 5 HLA-A2+ patients. We questioned whether this expansion specific to these two different cross-reactive responses would lead to alterations in the IAV-M158, EBV-BRLF1109 and -BMLF1280 TCR repertoires from before and during acute IAV infection. Using staining with VB mAb we found that T cell responses generated to these epitopes became surprisingly more polyclonal, with the sharing of Vb between M1, BMLF1 and BRLF1 populations which is not seen in healthy donor. Furthermore, by using next generation sequencing and single-cell analysis of TCRα and TCRβ repertoire of tetramer sorted IAV-M1 cells we showed dramatic changes in specific clonotype usage during acute IAV infection compared to before infection. In summary, these changes in TCR repertoire during acute symptomatic IAV infection suggest that during severe infection there is a preferential expansion of highly diverse cross-reactive responses between IAV and the persistent virus, EBV, which leads to permanent changes in TCR repertoires to both of these two viruses (NIH AI049320 and NIH AI109858).

Unreliable usage of a single influenza virus IgM antibody assay in influenza-like illness: A retrospective study of the 2016-2018 flu epidemic.

We retrospectively analyzed serum IgM antibodies (Abs) to influenza viruses from two tertiary hospitals in Beijing from December 2016 to February 2018. Samples from 36,792 patients, aged 0–98 years, were collected and tested. Among the patients, 923 children from two winter flu seasons were assayed with both antigens and IgM Abs to Flu A and Flu B and assigned as paired groups. Another 2,340 adults and 1,978 children with only antigen tested in the 2016 and 2017 winter flu seasons were named as unpaired groups. IgM Abs-positivity rates in children were 0.80% and 36.57% for Flu A and Flu B, respectively, peaking at 4–5 years of age. For adults, the Flu A and Flu B IgM Abs-positivity rates were 10.34% and 21.49%, respectively, peaking at 18–35 years of age. The trend of temporal distribution between the children and the adults was significantly correlated for IgM Abs to Flu B, but not for Flu A. Compared with unpaired groups, the detection rate of Flu A antigen was significantly higher than IgM Abs in children, whereas frequencies of IgM Abs were higher than antigen in adults. Incidence of Flu B antigen was sharply increased in 2017 winter than in the 2016 winter in both children and adults, but no concomitant increase was observed in IgM Abs to Flu B. For paired children groups, incidence of Flu B antigen in the 2017 flu season was significantly higher than that in the 2016 flu season; in contrast, positive rates of IgM Abs in the 2017 flu season were even lower than those in 2016. Considering antigen detection may reflect the Flu A/Flu B epidemic, our results indicate single-assayed IgM Abs were less effective in the diagnosis of acute influenza virus infection, and the use of this assay for epidemiology evaluations was not supported by these findings.

Nmp4-deficiency protects mice from influenza A virus infection

Abstract Severe influenza A virus infection typically triggers excessive and detrimental lung inflammation with massive cell infiltration and hyper-production of cytokines and chemokines. Discovering research models to alleviate lung damage without compromising viral control is critical. Here we identify that the lack of transcription factor-nuclear matrix protein 4 (Nmp4) protects mice from acute influenza virus infection by modulating the early stage inflammation. Upon infection, Nmp4-deficient mice lose only 5% body weight compared to 20% for the wild type (WT) mice. Furthermore, Nmp4-deficiency significantly reduces the recruitment of neutrophils and macrophages in the bronchoalveolar lavage fluid and lung parenchyma. Consistent with the fewer innate cells in the airways, influenza infected Nmp4-deficient mice have significantly decreased expression of cytokines and chemokines, such as IL-1β, TNF, IFN-γ, and MCP-1. However, Nmp4-deficient mice show similar viral control and adaptive immune responses to the WT mice with comparable virus specific CD8 T cells, CD4 T cells and anti-viral antibodies. Taken together, our data suggest that loss of Nmp4 protects mice from immune-driven lung damage without affecting viral clearance and may serve as a therapeutic target for severe influenza infection.

Vaccine-induced antigen-specific regulatory T cells attenuate the antiviral immunity against acute influenza virus infection.

Peptide-based T cell vaccines targeting the conserved epitopes of influenza virus can provide cross-protection against distantly related strains, but they are generally not immunogenic. Foreign antigen-specific regulatory T (Treg) cells are induced under subimmunogenic conditions peripherally, although their development and role in vaccine-mediated antiviral immunity is unclear. Here, we demonstrated primary vaccination with peptides alone significantly induced antigen-specific Foxp3+ Treg cells, which were further expanded by repeated vaccination with unadjuvanted peptides. Certain adjuvants, including CpG, suppressed the induction and expansion of antigen-specific Treg cells by peptide vaccination. Interestingly, secondary influenza virus infection significantly increased the frequency of preexisting antigen-specific Treg cells, although primary infection barely induced them. Importantly, specific depletion of vaccine-induced antigen-specific Treg cells promoted influenza viral clearance, indicating their inhibitory role in vivo. Immunization with CpG-adjuvanted peptides by the subcutaneous prime-intranasal-boost strategy restricted the recruitment and accumulation of antigen-specific Treg cells in lung, and stimulated robust T cell immunity. Finally, subcutaneous prime-intranasal-boost immunization with CpG-adjuvanted peptides or whole-inactivated influenza vaccines protected mice from heterosubtypic influenza virus infection. In conclusion, antigen-specific Treg cells induced by peptide vaccines attenuate the antiviral immunity against influenza virus infection. CpG-adjuvanted peptide vaccines provide heterosubtypic influenza protection probably by inhibiting Treg development and enhancing T cell immunity.

Influenza A virus (IAV) infection in humans leads to expansion of highly diverse CD8 T cell repertoires cross-reactive with Epstein Barr virus (EBV)

Abstract The competence of T cell responses predominantly depends on how efficient T cell receptors (TCRs) are at recognizing antigenic epitopes. We show here that during acute severely symptomatic IAV infection there was an expansion of IAV-M1/EBV-BRLF1 and IAV-M1/EBV-BMLF1 double-tetramer+ cells directly ex-vivo in 5 HLA-A2+ patients. We questioned whether this expansion specific to these two different cross-reactive responses would lead to alterations in the IAV-M158, EBV-BRLF1109 and -BMLF1280 TCR repertoires from before and during acute IAV infection. Using staining with VB mAb we found that T cell responses generated to these epitopes became surprisingly more polyclonal, with the sharing of Vb between M1, BMLF1 and BRLF1 populations which is not seen in healthy donor. Furthermore, by using next generation sequencing and single-cell analysis of TCRα and TCRβ repertoire of tetramer sorted IAV-M1 cells we showed dramatic changes in specific clonotype usage during acute IAV infection compared to before infection. In summary, these changes in TCR repertoire during acute symptomatic IAV infection suggest that during severe infection there is a preferential expansion of highly diverse cross-reactive responses between IAV and the persistent virus, EBV, which leads to permanent changes in TCR repertoires to both of these two viruses (NIH AI049320 and NIH AI109858).

Suppression of vaccination-induced antigen-specific regulatory T cells enhances the antiviral immunity against influenza virus infection

Abstract T cell immunity targeting the conserved epitopes of influenza virus has the potential to provide cross-protection against distinctly-related strains. Peptide vaccines are an ideal strategy to induce specific antiviral T cell immunity, but subimmunogenic stimulation by peptide vaccines tend to induce regulatory T (Treg) cells. However, it remains unclear about the roles of vaccine-induced antigen-specific Treg cells in vivo. Here, we aimed to investigate how vaccine-induced antigen-specific Treg cells respond during acute influenza virus infection, and the effects of adjuvants on them. By adoptive transfer experiments with OT-II TCR transgenic T cells, we found that the OVA OT-II peptide vaccine induced Treg cells and secondary vaccination further expanded them. Infection with influenza virus containing the OVA OT-II peptide also drove the expansion of pre-existing vaccine-induced Treg cells. Importantly, specific depletion of vaccine-induced antigen-specific Treg cells enhanced the antiviral immunity. Vaccination combined with adjuvants, especially with CpG, suppressed the development of vaccine-induced antigen-specific Treg cells. Finally, CpG-adjuvanted OVA protein or whole inactivated influenza vaccine by the subcutaneous-prime-intranasal-boost strategy reduced the ratio of antigen-specific Treg cells in lung and protected mice from the heterosubtypic influenza infection. In conclusion, unadjuvanted peptide vaccines promoted antigen-specific Treg cells, which were further expanded upon acute influenza infection. The CpG adjuvant restricted the development of vaccine-induced antigen-specific Treg cells, and enhanced the antiviral T cell immunity against heterosubtypic influenza infection.