Polyclonal but not monoclonal circulating memory CD4+ T cells attenuate the severity of Staphylococcus aureus bacteremia.

Staphylococcus aureus bacteremia causes significant morbidity and mortality. Treatment of staphylococcal infections is hindered by widespread antibiotic resistance, and attempts to develop an S. aureus vaccine have failed. Improved S. aureus treatment and infection prevention options require a deeper understanding of the correlates of protective immunity. CD4+ T cells have been identified as key orchestrators in the defense against S. aureus, but uncertainties persist regarding the subset, polarity, and breadth of the memory CD4+ T-cell pool required for protection. Here, using a mouse model of systemic S. aureus infection, we discovered that the breadth of bacterium-specific memory CD4+ T-cell pool is a critical factor for protective immunity against invasive S. aureus infections. Seeding mice with a monoclonal bacterium-specific circulating memory CD4+ T-cell population failed to protect against systemic S. aureus infection; however, the introduction of a polyclonal and polyfunctional memory CD4+ T-cell pool significantly reduced the bacterial burden. Our findings support the development of a multi-epitope T-cell-based S. aureus vaccine, as a strategy to mitigate the severity of S. aureus bacteremia.

CD8+ T-cell responses towards conserved influenza B virus epitopes across anatomical sites and age.

Influenza B viruses (IBVs) cause substantive morbidity and mortality, and yet immunity towards IBVs remains understudied. CD8+ T-cells provide broadly cross-reactive immunity and alleviate disease severity by recognizing conserved epitopes. Despite the IBV burden, only 18 IBV-specific T-cell epitopes restricted by 5 HLAs have been identified currently. A broader array of conserved IBV T-cell epitopes is needed to develop effective cross-reactive T-cell based IBV vaccines. Here we identify 9 highly conserved IBV CD8+ T-cell epitopes restricted to HLA-B*07:02, HLA-B*08:01 and HLA-B*35:01. Memory IBV-specific tetramer+CD8+ T-cells are present within blood and tissues. Frequencies of IBV-specific CD8+ T-cells decline with age, but maintain a central memory phenotype. HLA-B*07:02 and HLA-B*08:01-restricted NP30-38 epitope-specific T-cells have distinct T-cell receptor repertoires. We provide structural basis for the IBV HLA-B*07:02-restricted NS1196-206 (11-mer) and HLA-B*07:02-restricted NP30-38 epitope presentation. Our study increases the number of IBV CD8+ T-cell epitopes, and defines IBV-specific CD8+ T-cells at cellular and molecular levels, across tissues and age.

Prior infection with unrelated neurotropic virus exacerbates influenza disease and impairs lung T cell responses.

Immunity to infectious diseases is predominantly studied by measuring immune responses towards a single pathogen, although co-infections are common. In-depth mechanisms on how co-infections impact anti-viral immunity are lacking, but are highly relevant to treatment and prevention. We established a mouse model of co-infection with unrelated viruses, influenza A (IAV) and Semliki Forest virus (SFV), causing disease in different organ systems. SFV infection eight days before IAV infection results in prolonged IAV replication, elevated cytokine/chemokine levels and exacerbated lung pathology. This is associated with impaired lung IAV-specific CD8+ T cell responses, stemming from suboptimal CD8+ T cell activation and proliferation in draining lymph nodes, and dendritic cell paralysis. Prior SFV infection leads to increased blood brain barrier permeability and presence of IAV RNA in brain, associated with increased trafficking of IAV-specific CD8+ T cells and establishment of long-term tissue-resident memory. Relative to lung IAV-specific CD8+ T cells, brain memory IAV-specific CD8+ T cells have increased TCR repertoire diversity within immunodominant DbNP366+CD8+ and DbPA224+CD8+ responses, featuring suboptimal TCR clonotypes. Overall, our study demonstrates that infection with an unrelated neurotropic virus perturbs IAV-specific immune responses and exacerbates IAV disease. Our work provides key insights into therapy and vaccine regimens directed against unrelated pathogens.

Intranasal Epitope-Polymer Vaccine Lodges Resident Memory T Cells Protecting Against Influenza Virus.

Intranasal vaccines, unlike injectable vaccines, boost immunity along the respiratory tract; this can significantly limit respiratory virus replication and shedding. There remains a need to develop mucosal adjuvants and vaccine delivery systems that are both safe and effective following intranasal administration. Here, biopolymer particles (BP) densely coated with repeats of MHC class I restricted immunodominant epitopes derived from influenza A virus namely NP366, a nucleoprotein-derived epitope and PA224, a polymerase acidic subunit derived epitope, are bioengineered. These BP-NP366/PA224 can be manufactured at a high yield and are obtained at ≈93% purity, exhibiting ambient-temperature stability. Immunological characterization includes comparing systemic and mucosal immune responses mounted following intramuscular or intranasal immunization. Immunization with BP-NP366/PA224 without adjuvant triggers influenza-specific CD8+ T cell priming and memory CD8+ T cell development. Co-delivery with the adjuvant poly(I:C) significantly boosts the size and functionality of the influenza-specific pulmonary resident memory CD8+ T cell pool. Intranasal, but not intramuscular delivery of BP-NP366/PA224 with poly(I:C), provides protection against influenza virus challenge. Overall, the BP approach demonstrates as a suitable antigen formulation for intranasal delivery toward induction of systemic protective T cell responses against influenza virus.

Single-cycle influenza virus vaccine generates lung CD8+ Trm that cross-react against viral variants and subvert virus escape mutants.

Influenza virus-specific tissue-resident memory (Trm) CD8+ T cells located along the respiratory tract provide cross-strain protection against a breadth of influenza viruses. We show that immunization with a single-cycle influenza virus vaccine candidate (S-FLU) results in the deposition of influenza virus nucleoprotein (NP)-specific CD8+ Trm along the respiratory tract that were more cross-reactive against viral variants and less likely to drive the development of cytotoxic T lymphocyte (CTL) escape mutants, as compared to the lung memory NP-specific CD8+ T cell pool established following influenza infection. This immune profile was linked to the limited inflammatory response evoked by S-FLU vaccination, which increased TCR repertoire diversity within the memory CD8+ T cell compartment. Cumulatively, this work shows that S-FLU vaccination evokes a clonally diverse, cross-reactive memory CD8+ T cell pool, which protects against severe disease without driving the virus to rapidly evolve and escape, and thus represents an attractive vaccine for use against rapidly mutating influenza viruses.

Vaccine-induced inflammation and inflammatory monocytes promote CD4+ T cell-dependent immunity against murine salmonellosis.

Prior infection can generate protective immunity against subsequent infection, although the efficacy of such immunity can vary considerably. Live-attenuated vaccines (LAVs) are one of the most effective methods for mimicking this natural process, and analysis of their efficacy has proven instrumental in the identification of protective immune mechanisms. Here, we address the question of what makes a LAV efficacious by characterising immune responses to a LAV, termed TAS2010, which is highly protective (80-90%) against lethal murine salmonellosis, in comparison with a moderately protective (40-50%) LAV, BRD509. Mice vaccinated with TAS2010 developed immunity systemically and were protected against gut-associated virulent infection in a CD4+ T cell-dependent manner. TAS2010-vaccinated mice showed increased activation of Th1 responses compared with their BRD509-vaccinated counterparts, leading to increased Th1 memory populations in both lymphoid and non-lymphoid organs. The optimal development of Th1-driven immunity was closely correlated with the activation of CD11b+Ly6GnegLy6Chi inflammatory monocytes (IMs), the activation of which can be modulated proportionally by bacterial load in vivo. Upon vaccination with the LAV, IMs expressed T cell chemoattractant CXCL9 that attracted CD4+ T cells to the foci of infection, where IMs also served as a potent source of antigen presentation and Th1-promoting cytokine IL-12. The expression of MHC-II in IMs was rapidly upregulated following vaccination and then maintained at an elevated level in immune mice, suggesting IMs may have a role in sustained antigen stimulation. Our findings present a longitudinal analysis of CD4+ T cell development post-vaccination with an intracellular bacterial LAV, and highlight the benefit of inflammation in the development of Th1 immunity. Future studies focusing on the induction of IMs may reveal key strategies for improving vaccine-induced T cell immunity.

Cutting Edge: High-Dose Live Attenuated Influenza Vaccines Elicit Pulmonary Tissue-Resident Memory CD8+ T Cells in the Face of Pre-Existing Humoral Immunity.

In this study, we investigated how pre-existing Ab immunity to influenza virus established from prior immunizations affects the development of CD8+ T cell responses evoked after vaccination with a live attenuated vaccine. Using a mouse model and a panel of live attenuated influenza virus vaccine candidates (cold adapted and single cycle), we show that pre-existing influenza-specific Abs directed against the vaccine backbone attenuate the size and quality of the vaccine-induced CD8+ T cell response. Importantly, we show that increasing the vaccine dose can overcome this impediment, resulting in improved vaccine-induced circulating and tissue-resident memory CD8+ T cell responses, which were protective against heterologous influenza challenge. Thus, the reduced size and quality of the T cell response elicited by a live attenuated influenza virus vaccine imparted by the influenza-specific Ab landscape of the vaccinee can be overcome by increasing vaccine dose.

Mouse Mx1 Inhibits Herpes Simplex Virus Type 1 Genomic Replication and Late Gene Expression In Vitro and Prevents Lesion Formation in the Mouse Zosteriform Model.

Myxovirus resistance (Mx) proteins are dynamin-like GTPases that are inducible by interferons (IFNs) following virus infections. Most studies investigating Mx proteins have focused on their activity against influenza A viruses (IAV), although emerging evidence suggests that some Mx proteins may exhibit broader antiviral activity. Herein, we demonstrate that in addition to IAV, overexpression of mouse Mx1 (mMx1), but not mMx2, resulted in potent inhibition of growth of the human alphaherpesviruses herpes simplex virus 1 (HSV-1) and HSV-2, whereas neither inhibited the mouse betaherpesvirus murine cytomegalovirus (MCMV) in vitro. IFN induction of a functional endogenous mMx1 in primary mouse fibroblasts ex vivo was also associated with inhibition of HSV-1 growth. Using an in vitro overexpression approach, we demonstrate that mutations that result in redistribution of mMx1 from the nucleus to the cytoplasm or in loss of its combined GTP binding and GTPase activity also abrogated its ability to inhibit HSV-1 growth. Overexpressed mMx1 did not inhibit early HSV-1 gene expression but was shown to inhibit both replication of the HSV-1 genome as well as subsequent late gene expression. In a mouse model of cutaneous HSV-1 infection, mice expressing a functional endogenous mMx1 showed significant reductions in the severity of skin lesions as well as reduced HSV-1 titers in both the skin and dorsal root ganglia (DRG). Together, these data demonstrate that mMx1 mediates potent antiviral activity against human alphaherpesviruses by blocking replication of the viral genome and subsequent steps in virus replication. Moreover, endogenous mMx1 potently inhibited pathogenesis in the zosteriform mouse model of HSV-1 infection. IMPORTANCE While a number of studies have demonstrated that human Mx proteins can inhibit particular herpesviruses in vitro, we are the first to report the antiviral activity of mouse Mx1 (mMx1) against alphaherpesviruses both in vitro and in vivo. We demonstrate that both overexpressed mMx1 and endogenous mMx1 potently restrict HSV-1 growth in vitro. mMx1-mediated inhibition of HSV-1 was not associated with inhibition of virus entry and/or import of the viral genome into the nucleus, but rather with inhibition of HSV-1 genomic replication as well as subsequent late gene expression. Therefore, inhibition of human alphaherpesviruses by mMx1 occurs by a mechanism that is distinct from that reported for human Mx proteins against herpesviruses. Importantly, we also provide evidence that expression of a functional endogenous mMx1 can limit HSV-1 pathogenesis in a mouse model of infection.

SARS-CoV-2 infection results in immune responses in the respiratory tract and peripheral blood that suggest mechanisms of disease severity.

Respiratory tract infection with SARS-CoV-2 results in varying immunopathology underlying COVID-19. We examine cellular, humoral and cytokine responses covering 382 immune components in longitudinal blood and respiratory samples from hospitalized COVID-19 patients. SARS-CoV-2-specific IgM, IgG, IgA are detected in respiratory tract and blood, however, receptor-binding domain (RBD)-specific IgM and IgG seroconversion is enhanced in respiratory specimens. SARS-CoV-2 neutralization activity in respiratory samples correlates with RBD-specific IgM and IgG levels. Cytokines/chemokines vary between respiratory samples and plasma, indicating that inflammation should be assessed in respiratory specimens to understand immunopathology. IFN-α2 and IL-12p70 in endotracheal aspirate and neutralization in sputum negatively correlate with duration of hospital stay. Diverse immune subsets are detected in respiratory samples, dominated by neutrophils. Importantly, dexamethasone treatment does not affect humoral responses in blood of COVID-19 patients. Our study unveils differential immune responses between respiratory samples and blood, and shows how drug therapy affects immune responses during COVID-19.

Staphylococcus aureus specific lung resident memory CD4+ Th1 cells attenuate the severity of influenza virus induced secondary bacterial pneumonia.

Staphylococcus aureus is a major cause of severe pulmonary infections. The evolution of multi-drug resistant strains limits antibiotic treatment options. To date, all candidate vaccines tested have failed, highlighting the need for an increased understanding of the immunological requirements for effective S. aureus immunity. Using an S. aureus strain engineered to express a trackable CD4+ T cell epitope and a murine model of S. aureus pneumonia, we show strategies that lodge Th1 polarised bacterium specific CD4+ tissue resident memory T cells (Trm) in the lung can significantly attenuate the severity of S. aureus pneumonia. This contrasts natural infection of mice that fails to lodge CD4+ Trm cells along the respiratory tract or provide protection against re-infection, despite initially generating Th17 bacterium specific CD4+ T cell responses. Interestingly, lack of CD4+ Trm formation after natural infection in mice appears to be reflected in humans, where the frequency of S. aureus reactive CD4+ Trm cells in lung tissue is also low. Our findings reveal the protective capacity of S. aureus specific respiratory tract CD4+ Th1 polarised Trm cells and highlight the potential for targeting these cells in vaccines that aim to prevent the development of S. aureus pneumonia.

Tissue resident memory T cells in the respiratory tract.

Owing to their capacity to rapidly spread across the population, airborne pathogens represent a significant risk to global health. Indeed, several of the past major global pandemics have been instigated by respiratory pathogens. A greater understanding of the immune cells tasked with protecting the airways from infection will allow for the development of strategies that curb the spread and impact of these airborne diseases. A specific subset of memory T-cell resident in both the upper and lower respiratory tract, termed tissue-resident memory (Trm), have been shown to play an instrumental role in local immune responses against a wide breadth of both viral and bacterial infections. In this review, we discuss factors that influence respiratory tract Trm development, longevity, and immune surveillance and explore vaccination regimes that harness these cells, such approaches represent exciting new strategies that may be utilized to tackle the next global pandemic.

CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity.

To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8+ T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8+ T cells directed toward subdominant epitopes (B7/N257, A2/S269, and A24/S1,208) CD8+ T cells specific for the immunodominant B7/N105 epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8+ T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαß repertoires and promiscuous αß-TCR pairing within B7/N105+CD8+ T cells. Our study demonstrates high naive precursor frequency and TCRαß diversity within immunodominant B7/N105-specific CD8+ T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.

CD8+ T cell landscape in Indigenous and non-Indigenous people restricted by influenza mortality-associated HLA-A*24:02 allomorph.

Indigenous people worldwide are at high risk of developing severe influenza disease. HLA-A*24:02 allele, highly prevalent in Indigenous populations, is associated with influenza-induced mortality, although the basis for this association is unclear. Here, we define CD8+ T-cell immune landscapes against influenza A (IAV) and B (IBV) viruses in HLA-A*24:02-expressing Indigenous and non-Indigenous individuals, human tissues, influenza-infected patients and HLA-A*24:02-transgenic mice. We identify immunodominant protective CD8+ T-cell epitopes, one towards IAV and six towards IBV, with A24/PB2550-558-specific CD8+ T cells being cross-reactive between IAV and IBV. Memory CD8+ T cells towards these specificities are present in blood (CD27+CD45RA- phenotype) and tissues (CD103+CD69+ phenotype) of healthy individuals, and effector CD27-CD45RA-PD-1+CD38+CD8+ T cells in IAV/IBV patients. Our data show influenza-specific CD8+ T-cell responses in Indigenous Australians, and advocate for T-cell-mediated vaccines that target and boost the breadth of IAV/IBV-specific CD8+ T cells to protect high-risk HLA-A*24:02-expressing Indigenous and non-Indigenous populations from severe influenza disease.

Influenza, but not SARS-CoV-2, infection induces a rapid interferon response that wanes with age and diminished tissue-resident memory CD8+ T cells.

Older individuals exhibit a diminished ability to respond to and clear respiratory pathogens and, as such, experience a higher rate of lung infections with a higher mortality rate. It is unclear why respiratory pathogens impact older people disproportionately. Using human lung tissue from donors aged 22-68 years, we assessed how the immune cell landscape in lungs changes throughout life and investigated how these immune cells respond following in vitro exposure to influenza virus and SARS-CoV-2, two clinically relevant respiratory viruses. While the frequency of most immune cell subsets profiled in the human lung remained stable with age, memory CD8+ T cells declined, with the tissue-resident memory (Trm) CD8+ T-cell subset being most susceptible to age-associated attrition. Infection of lung tissue with influenza virus resulted in an age-associated attenuation in the antiviral immune response, with aged donors producing less type I interferon (IFN), GM-CSF and IFNγ, the latter correlated with a reduction of IFNγ-producing memory CD8+ T cells. In contrast, irrespective of donor age, exposure of human lung cells to SARS-CoV-2, a pathogen for which all donors were immunologically naïve, did not trigger activation of local immune cells and did not result in the induction of an early IFN response. Our findings show that the attrition of tissue-bound pathogen-specific Trm in the lung that occurs with advanced age, or their absence in immunologically naïve individuals, results in a diminished early antiviral immune response which creates a window of opportunity for respiratory pathogens to gain a greater foothold.

RNF41 regulates the damage recognition receptor Clec9A and antigen cross-presentation in mouse dendritic cells.

The dendritic cell receptor Clec9A facilitates processing of dead cell-derived antigens for cross-presentation and the induction of effective CD8+ T cell immune responses. Here, we show that this process is regulated by E3 ubiquitin ligase RNF41 and define a new ubiquitin-mediated mechanism for regulation of Clec9A, reflecting the unique properties of Clec9A as a receptor specialized for delivery of antigens for cross-presentation. We reveal RNF41 is a negative regulator of Clec9A and the cross-presentation of dead cell-derived antigens by mouse dendritic cells. Intriguingly, RNF41 regulates the downstream fate of Clec9A by directly binding and ubiquitinating the extracellular domains of Clec9A. At steady-state, RNF41 ubiquitination of Clec9A facilitates interactions with ER-associated proteins and degradation machinery to control Clec9A levels. However, Clec9A interactions are altered following dead cell uptake to favor antigen presentation. These findings provide important insights into antigen cross-presentation and have implications for development of approaches to modulate immune responses.

Intranasal Delivery of a Chitosan-Hydrogel Vaccine Generates Nasal Tissue Resident Memory CD8+ T Cells That Are Protective against Influenza Virus Infection.

Rapid antigen clearance from the nasal mucosa is one of the major challenges in the development of intranasal vaccines. Here, we tested whether intranasal immunization with a chitosan-hydrogel vaccine, with in situ gelling properties, extended antigen retention time within the nasal mucosa. Intranasal immunization with a chitosan-hydrogel vaccine retained antigen within the upper respiratory tract (URT), while intranasal delivery of less viscous vaccines led to antigen accumulation within the lower airways. Interestingly, sustained antigen retention within the URT following chitosan-hydrogel vaccination boosted the number of vaccine-specific, tissue resident memory (Trm) CD8+ T cells that developed within the nasal mucosa. Mice immunized with a chitosan-hydrogel vaccine loaded with influenza virus peptides developed a large pool of influenza-specific CD8+ nasal Trm and these cells were highly protective during an influenza challenge. Our results describe an effective vaccine formulation that can be utilized to boost local immunity in the nasal mucosa.

Unresponsiveness to inhaled antigen is governed by conventional dendritic cells and overridden during infection by monocytes.

The nasal-associated lymphoid tissues (NALTs) are mucosal-associated lymphoid organs embedded in the submucosa of the nasal passage. NALTs represent a known site for the deposition of inhaled antigens, but little is known of the mechanisms involved in the induction of immunity within this lymphoid tissue. We find that during the steady state, conventional dendritic cells (cDCs) within the NALTs suppress T cell responses. These cDCs, which are also prevalent within human NALTs (tonsils/adenoids), express a unique transcriptional profile and inhibit T cell proliferation via contact-independent mechanisms that can be diminished by blocking the actions of reactive oxygen species and prostaglandin E2 Although the prevention of unrestrained immune activation to inhaled antigens appears to be the default function of NALT cDCs, inflammation after localized virus infection recruited monocyte-derived DCs (moDCs) to this region, which diluted out the suppressive DC pool, and permitted local T cell priming. Accommodating for inflammation-induced temporal changes in NALT DC composition and function, we developed an intranasal vaccine delivery system that coupled the recruitment of moDCs with the sustained release of antigen into the NALTs, and we were able to substantially improve T cell responses after intranasal immunization. Thus, homeostasis and immunity to inhaled antigens is tuned by inflammatory signals that regulate the balance between conventional and moDC populations within the NALTs.

Suboptimal SARS-CoV-2-specific CD8+ T cell response associated with the prominent HLA-A*02:01 phenotype.

An improved understanding of human T cell-mediated immunity in COVID-19 is important for optimizing therapeutic and vaccine strategies. Experience with influenza shows that infection primes CD8+ T cell memory to peptides presented by common HLA types like HLA-A2, which enhances recovery and diminishes clinical severity upon reinfection. Stimulating peripheral blood mononuclear cells from COVID-19 convalescent patients with overlapping peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the clonal expansion of SARS-CoV-2-specific CD8+ and CD4+ T cells in vitro, with CD4+ T cells being robust. We identified two HLA-A*02:01-restricted SARS-CoV-2-specfic CD8+ T cell epitopes, A2/S269-277 and A2/Orf1ab3183-3191 Using peptide-HLA tetramer enrichment, direct ex vivo assessment of A2/S269+CD8+ and A2/Orf1ab3183+CD8+ populations indicated that A2/S269+CD8+ T cells were detected at comparable frequencies (∼1.3 × 10-5) in acute and convalescent HLA-A*02:01+ patients. These frequencies were higher than those found in uninfected HLA-A*02:01+ donors (∼2.5 × 10-6), but low when compared to frequencies for influenza-specific (A2/M158) and Epstein-Barr virus (EBV)-specific (A2/BMLF1280) (∼1.38 × 10-4) populations. Phenotyping A2/S269+CD8+ T cells from COVID-19 convalescents ex vivo showed that A2/S269+CD8+ T cells were predominantly negative for CD38, HLA-DR, PD-1, and CD71 activation markers, although the majority of total CD8+ T cells expressed granzymes and/or perforin. Furthermore, the bias toward naïve, stem cell memory and central memory A2/S269+CD8+ T cells rather than effector memory populations suggests that SARS-CoV-2 infection may be compromising CD8+ T cell activation. Priming with appropriate vaccines may thus be beneficial for optimizing CD8+ T cell immunity in COVID-19.

Airway Exosomes Released During Influenza Virus Infection Serve as a Key Component of the Antiviral Innate Immune Response.

Exosomes are extracellular vesicles secreted by cells that have an important biological function in intercellular communication by transferring biologically active proteins, lipids, and RNAs to neighboring or distant cells. While a role for exosomes in antimicrobial defense has recently emerged, currently very little is known regarding the nature and functional relevance of exosomes generated in vivo, particularly during an active viral infection. Here, we characterized exosomes released into the airways during influenza virus infection. We show that these vesicles dynamically change in protein composition over the course of infection, increasing expression of host proteins with known anti-influenza activity, and viral proteins with the potential to trigger host immune responses. We show that exosomes released into the airways during influenza virus infection trigger pulmonary inflammation and carry viral antigen that can be utilized by antigen presenting cells to drive the induction of a cellular immune response. Moreover, we show that attachment factors for influenza virus, namely α2,3 and α2,6-linked sialic acids, are present on the surface of airway exosomes and these vesicles have the ability to neutralize influenza virus, thereby preventing the virus from binding and entering target cells. These data reveal a novel role for airway exosomes in the antiviral innate immune defense against influenza virus infection.

Neutrophils play an ongoing role in preventing bacterial pneumonia by blocking the dissemination of Staphylococcus aureus from the upper to the lower airways.

Staphylococcus aureus is found in the nasal cavity of up to 30% of the human population. Persistent nasal carriage of S. aureus is a risk factor for influenza virus-induced secondary bacterial pneumonia. There is limited understanding of the factors that cause S. aureus to shift from the upper to the lower respiratory tract and convert from a commensal organism to an invasive pathogen. Here we show that neutrophils actively prevent S. aureus dissemination. Establishment of a mouse model of localized S. aureus nasal carriage revealed variations in the longevity of persistence of S. aureus isolates. Improved persistence within this site was associated with reduced nasal inflammation, less neutrophil egress into the airways and reduced neutrophil-bacteria association. Neutrophil depletion of mice with localized S. aureus nasal carriage triggered the development of an invasive S. aureus infection. Moreover, utilizing a model of influenza-induced staphylococcal pneumonia we showed that treatment with granulocyte-colony-stimulating factor, a potent enhancer of neutrophil number and function, significantly reduced bacterial loads in the lung and improved disease outcomes. These data reveal that neutrophils play an important and active role in confining S. aureus to the upper respiratory tract and highlight the use of approaches that improve neutrophil function as effective strategies to attenuate morbidity associated with staphylococcal pneumonia.