Antibiotics-Driven Gut Microbiome Perturbation Alters Immunity to Vaccines in Humans.

Emerging evidence indicates a central role for the microbiome in immunity. However, causal evidence in humans is sparse. Here, we administered broad-spectrum antibiotics to healthy adults prior and subsequent to seasonal influenza vaccination. Despite a 10,000-fold reduction in gut bacterial load and long-lasting diminution in bacterial diversity, antibody responses were not significantly affected. However, in a second trial of subjects with low pre-existing antibody titers, there was significant impairment in H1N1-specific neutralization and binding IgG1 and IgA responses. In addition, in both studies antibiotics treatment resulted in (1) enhanced inflammatory signatures (including AP-1/NR4A expression), observed previously in the elderly, and increased dendritic cell activation; (2) divergent metabolic trajectories, with a 1,000-fold reduction in serum secondary bile acids, which was highly correlated with AP-1/NR4A signaling and inflammasome activation. Multi-omics integration revealed significant associations between bacterial species and metabolic phenotypes, highlighting a key role for the microbiome in modulating human immunity.

m6A modification controls the innate immune response to infection by targeting type I interferons.

N6-methyladenosine (m6A) is the most common mRNA modification. Recent studies have revealed that depletion of m6A machinery leads to alterations in the propagation of diverse viruses. These effects were proposed to be mediated through dysregulated methylation of viral RNA. Here we show that following viral infection or stimulation of cells with an inactivated virus, deletion of the m6A 'writer' METTL3 or 'reader' YTHDF2 led to an increase in the induction of interferon-stimulated genes. Consequently, propagation of different viruses was suppressed in an interferon-signaling-dependent manner. Significantly, the mRNA of IFNB, the gene encoding the main cytokine that drives the type I interferon response, was m6A modified and was stabilized following repression of METTL3 or YTHDF2. Furthermore, we show that m6A-mediated regulation of interferon genes was conserved in mice. Together, our findings uncover the role m6A serves as a negative regulator of interferon response by dictating the fast turnover of interferon mRNAs and consequently facilitating viral propagation.

Mosaic nanoparticle display of diverse influenza virus hemagglutinins elicits broad B cell responses.

The present vaccine against influenza virus has the inevitable risk of antigenic discordance between the vaccine and the circulating strains, which diminishes vaccine efficacy. This necessitates new approaches that provide broader protection against influenza. Here we designed a vaccine using the hypervariable receptor-binding domain (RBD) of viral hemagglutinin displayed on a nanoparticle (np) able to elicit antibody responses that neutralize H1N1 influenza viruses spanning over 90 years. Co-display of RBDs from multiple strains across time, so that the adjacent RBDs are heterotypic, provides an avidity advantage to cross-reactive B cells. Immunization with the mosaic RBD-np elicited broader antibody responses than those induced by an admixture of nanoparticles encompassing the same set of RBDs as separate homotypic arrays. Furthermore, we identified a broadly neutralizing monoclonal antibody in a mouse immunized with mosaic RBD-np. The mosaic antigen array signifies a unique approach that subverts monotypic immunodominance and allows otherwise subdominant cross-reactive B cell responses to emerge.

Single-Cell Sequencing of Peripheral Mononuclear Cells Reveals Distinct Immune Response Landscapes of COVID-19 and Influenza Patients.

The coronavirus disease 2019 (COVID-19) pandemic poses a current world-wide public health threat. However, little is known about its hallmarks compared to other infectious diseases. Here, we report the single-cell transcriptional landscape of longitudinally collected peripheral blood mononuclear cells (PBMCs) in both COVID-19- and influenza A virus (IAV)-infected patients. We observed increase of plasma cells in both COVID-19 and IAV patients and XIAP associated factor 1 (XAF1)-, tumor necrosis factor (TNF)-, and FAS-induced T cell apoptosis in COVID-19 patients. Further analyses revealed distinct signaling pathways activated in COVID-19 (STAT1 and IRF3) versus IAV (STAT3 and NFκB) patients and substantial differences in the expression of key factors. These factors include relatively increase of interleukin (IL)6R and IL6ST expression in COVID-19 patients but similarly increased IL-6 concentrations compared to IAV patients, supporting the clinical observations of increased proinflammatory cytokines in COVID-19 patients. Thus, we provide the landscape of PBMCs and unveil distinct immune response pathways in COVID-19 and IAV patients.

Protective neutralizing influenza antibody response in the absence of T follicular helper cells.

Virus infection induces the development of T follicular helper (TFH) and T helper 1 (TH1) cells. Although TFH cells are important in anti-viral humoral immunity, the contribution of TH1 cells to a protective antibody response remains unknown. We found that IgG2 antibodies predominated in the response to vaccination with inactivated influenza A virus (IAV) and were responsible for protective immunity to lethal challenge with pathogenic H5N1 and pandemic H1N1 IAV strains, even in mice that lacked TFH cells and germinal centers. The cytokines interleukin-21 and interferon-γ, which are secreted from TH1 cells, were essential for the observed greater persistence and higher titers of IgG2 protective antibodies. Our results suggest that TH1 induction could be a promising strategy for producing effective neutralizing antibodies against emerging influenza viruses.

Adjuvanted influenza-H1N1 vaccination reveals lymphoid signatures of age-dependent early responses and of clinical adverse events.

Adjuvanted vaccines afford invaluable protection against disease, and the molecular and cellular changes they induce offer direct insight into human immunobiology. Here we show that within 24 h of receiving adjuvanted swine flu vaccine, healthy individuals made expansive, complex molecular and cellular responses that included overt lymphoid as well as myeloid contributions. Unexpectedly, this early response was subtly but significantly different in people older than ∼35 years. Wide-ranging adverse clinical events can seriously confound vaccine adoption, but whether there are immunological correlates of these is unknown. Here we identify a molecular signature of adverse events that was commonly associated with an existing B cell phenotype. Thus immunophenotypic variation among healthy humans may be manifest in complex pathophysiological responses.

T-bet Transcription Factor Promotes Antibody-Secreting Cell Differentiation by Limiting the Inflammatory Effects of IFN-γ on B Cells.

Although viral infections elicit robust interferon-γ (IFN-γ) and long-lived antibody-secreting cell (ASC) responses, the roles for IFN-γ and IFN-γ-induced transcription factors (TFs) in ASC development are unclear. We showed that B cell intrinsic expression of IFN-γR and the IFN-γ-induced TF T-bet were required for T-helper 1 cell-induced differentiation of B cells into ASCs. IFN-γR signaling induced Blimp1 expression in B cells but also initiated an inflammatory gene program that, if not restrained, prevented ASC formation. T-bet did not affect Blimp1 upregulation in IFN-γ-activated B cells but instead regulated chromatin accessibility within the Ifng and Ifngr2 loci and repressed the IFN-γ-induced inflammatory gene program. Consistent with this, B cell intrinsic T-bet was required for formation of long-lived ASCs and secondary ASCs following viral, but not nematode, infection. Therefore, T-bet facilitates differentiation of IFN-γ-activated inflammatory effector B cells into ASCs in the setting of IFN-γ-, but not IL-4-, induced inflammatory responses.

B Cells Are the Dominant Antigen-Presenting Cells that Activate Naive CD4+ T Cells upon Immunization with a Virus-Derived Nanoparticle Antigen.

B cells can present antigens to CD4+ T cells, but it is thought that dendritic cells (DCs) are the primary initiators of naive CD4+ T cell responses. Nanoparticles, including virus-like particles (VLPs), are attractive candidates as carriers for vaccines and drug delivery. Using RNA phage Qß-derived VLP (Qß-VLP) as a model antigen, we found that antigen-specific B cells were the dominant antigen-presenting cells that initiated naive CD4+ T cell activation. B cells were sufficient to induce T follicular helper cell development in the absence of DCs. Qß-specific B cells promoted CD4+ T cell proliferation and differentiation via cognate interactions and through Toll-like receptor signaling-mediated cytokine production. Antigen-specific B cells were also involved in initiating CD4+ T cell responses during immunization with inactivated influenza virus. These findings have implications for the rational design of nanoparticles as vaccine candidates, particularly for therapeutic vaccines that aim to break immune tolerance.

Quadrivalent influenza nanoparticle vaccines induce broad protection.

Influenza vaccines that confer broad and durable protection against diverse viral strains would have a major effect on global health, as they would lessen the need for annual vaccine reformulation and immunization1. Here we show that computationally designed, two-component nanoparticle immunogens2 induce potently neutralizing and broadly protective antibody responses against a wide variety of influenza viruses. The nanoparticle immunogens contain 20 haemagglutinin glycoprotein trimers in an ordered array, and their assembly in vitro enables the precisely controlled co-display of multiple distinct haemagglutinin proteins in defined ratios. Nanoparticle immunogens that co-display the four haemagglutinins of licensed quadrivalent influenza vaccines elicited antibody responses in several animal models against vaccine-matched strains that were equivalent to or better than commercial quadrivalent influenza vaccines, and simultaneously induced broadly protective antibody responses to heterologous viruses by targeting the subdominant yet conserved haemagglutinin stem. The combination of potent receptor-blocking and cross-reactive stem-directed antibodies induced by the nanoparticle immunogens makes them attractive candidates for a supraseasonal influenza vaccine candidate with the potential to replace conventional seasonal vaccines3.

Single-cell analysis reveals lasting immunological consequences of influenza infection and respiratory immunization in the pig lung.

The pig is a natural host for influenza viruses and integrally involved in virus evolution through interspecies transmissions between humans and swine. Swine have many physiological, anatomical, and immunological similarities to humans, and are an excellent model for human influenza. Here, we employed single cell RNA-sequencing (scRNA-seq) and flow cytometry to characterize the major leukocyte subsets in bronchoalveolar lavage (BAL), twenty-one days after H1N1pdm09 infection or respiratory immunization with an adenoviral vector vaccine expressing hemagglutinin and nucleoprotein with or without IL-1ß. Mapping scRNA-seq clusters from BAL onto those previously described in peripheral blood facilitated annotation and highlighted differences between tissue resident and circulating immune cells. ScRNA-seq data and functional assays revealed lasting impacts of immune challenge on BAL populations. First, mucosal administration of IL-1ß reduced the number of functionally active Treg cells. Second, influenza infection upregulated IFI6 in BAL cells and decreased their susceptibility to virus replication in vitro. Our data provide a reference map of porcine BAL cells and reveal lasting immunological consequences of influenza infection and respiratory immunization in a highly relevant large animal model for respiratory virus infection.

Evaluation of a novel intramuscular prime/intranasal boost vaccination strategy against influenza in the pig model.

Live-attenuated influenza vaccines (LAIV) offer advantages over the commonly used inactivated split influenza vaccines. However, finding the optimal balance between sufficient attenuation and immunogenicity has remained a challenge. We recently developed an alternative LAIV based on the 2009 pandemic H1N1 virus with a truncated NS1 protein and lacking PA-X protein expression (NS1(1-126)-ΔPAX). This virus showed a blunted replication and elicited a strong innate immune response. In the present study, we evaluated the efficacy of this vaccine candidate in the porcine animal model as a pertinent in vivo system. Immunization of pigs via the nasal route with the novel NS1(1-126)-ΔPAX LAIV did not cause disease and elicited a strong mucosal immune response that completely blocked replication of the homologous challenge virus in the respiratory tract. However, we observed prolonged shedding of our vaccine candidate from the upper respiratory tract. To improve LAIV safety, we developed a novel prime/boost vaccination strategy combining primary intramuscular immunization with a haemagglutinin-encoding propagation-defective vesicular stomatitis virus (VSV) replicon, followed by a secondary immunization with the NS1(1-126)-ΔPAX LAIV via the nasal route. This two-step immunization procedure significantly reduced LAIV shedding, increased the production of specific serum IgG, neutralizing antibodies, and Th1 memory cells, and resulted in sterilizing immunity against homologous virus challenge. In conclusion, our novel intramuscular prime/intranasal boost regimen interferes with virus shedding and transmission, a feature that will help combat influenza epidemics and pandemics.

A chimeric mRNA vaccine of S-RBD with HA conferring broad protection against influenza and COVID-19 variants.

Influenza and coronavirus disease 2019 (COVID-19) represent two respiratory diseases that have significantly impacted global health, resulting in substantial disease burden and mortality. An optimal solution would be a combined vaccine capable of addressing both diseases, thereby obviating the need for multiple vaccinations. Previously, we conceived a chimeric protein subunit vaccine targeting both influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), utilizing the receptor binding domain of spike protein (S-RBD) and the stalk region of hemagglutinin protein (HA-stalk) components. By integrating the S-RBD from the SARS-CoV-2 Delta variant with the headless hemagglutinin (HA) from H1N1 influenza virus, we constructed stable trimeric structures that remain accessible to neutralizing antibodies. This vaccine has demonstrated its potential by conferring protection against a spectrum of strains in mouse models. In this study, we designed an mRNA vaccine candidate encoding the chimeric antigen. The resultant humoral and cellular immune responses were meticulously evaluated in mouse models. Furthermore, the protective efficacy of the vaccine was rigorously examined through challenges with either homologous or heterologous influenza viruses or SARS-CoV-2 strains. Our findings reveal that the mRNA vaccine exhibited robust immunogenicity, engendering high and sustained levels of neutralizing antibodies accompanied by robust and persistent cellular immunity. Notably, this vaccine effectively afforded complete protection to mice against H1N1 or heterosubtypic H5N8 subtypes, as well as the SARS-CoV-2 Delta and Omicron BA.2 variants. Additionally, our mRNA vaccine design can be easily adapted from Delta RBD to Omicron RBD antigens, providing protection against emerging variants. The development of two-in-one vaccine targeting both influenza and COVID-19, incorporating the mRNA platform, may provide a versatile approach to combating future pandemics.

The metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells.

Interleukin 15 (IL-15) controls both the homeostasis and the peripheral activation of natural killer (NK) cells. The molecular basis for this duality of action remains unknown. Here we found that the metabolic checkpoint kinase mTOR was activated and boosted bioenergetic metabolism after exposure of NK cells to high concentrations of IL-15, whereas low doses of IL-15 triggered only phosphorylation of the transcription factor STAT5. mTOR stimulated the growth and nutrient uptake of NK cells and positively fed back on the receptor for IL-15. This process was essential for sustaining NK cell proliferation during development and the acquisition of cytolytic potential during inflammation or viral infection. The mTORC1 inhibitor rapamycin inhibited NK cell cytotoxicity both in mice and humans; this probably contributes to the immunosuppressive activity of this drug in different clinical settings.

Oral co-administration of Lactiplantibacillus plantarum 16 and Lacticaseibacillus rhamnosus P118 improves host defense against influenza A virus infection.

Influenza is an important zoonotic disease that persistently threatens global public health. While it is widely acknowledged that probiotics can modulate the host response to protect the host against infectious disease, the prophylactic efficacy on respiratory viral infection and the detailed mechanism remains elusive. Lactobacillus, the most commonly used probiotic widely applied in food production, has garnered significant attention. In our study utilizing both C57BL/6 and BALB/c mouse models, we explored the protective effect against two strains of influenza virus, A/Mink/China/01/2014(H9N2) and A/California/04/2009(H1N1), through the administration of Lactiplantibacillus plantarum strain 16 (L. plantarum 16) and Lacticaseibacillus rhamnosus strain P118 (L. rhamnosus P118), aiming to identify robust probiotic strains with antiviral properties. Our findings indicate that administering L. plantarum 16 or L. rhamnosus P118 alone does not provide sufficient protection against influenza. However, the co-administration of L. plantarum 16 and L. rhamnosus P118 dramatically reduces viral titers in the respiratory tract and lung, thereby markedly alleviating the clinical symptoms, improving prognosis, and reducing mortality. The mechanisms underlying this effect involve the modulation of host gut microbiota and metabolism through the co-administration of L. plantarum 16 and L. rhamnosus P118, resulting in enrichment of Firmicutes and enhancement of phenylalanine-related metabolism, ultimately leading to an augmentation of the antiviral immune response. Notably, we identified that the circulating metabolic molecule 2-Hydroxycinnamic acid plays a significant role in combating influenza. Our data suggest the potential utility of L. plantarum 16 and L. rhamnosus P118 two-bacterium or 2-Hydroxycinnamic acid in preventing influenza.IMPORTANCEVaccination represents the most optimal strategy to control influenza. Nevertheless, influenza viruses constantly evolve due to antigenic drift and shift, leading to the need for regular updates on influenza vaccines. Additionally, vaccination failure poses significant challenges to influenza prevention. Therefore, it is essential and beneficial to identify novel or universal antiviral measures to protect against influenza. While cumulative data suggest that probiotics offer protection against infectious diseases, the specific mechanisms, such as the effective metabolites or components, remain largely unknown. Our research discovered the capacity of combinational two-bacterium Lactiplantibacillus plantarum 16 and Lacticaseibacillus rhamnosus P118 to fight against influenza infection in a mouse model. The protection may occur through modulating the host's gut microbiota and metabolism, further influencing the host's antiviral immune response. Notably, we have identified a novel metabolic molecule, 2-Hydroxycinnamic acid, capable of enhancing antiviral response and restricting viral replication in vivo.

Secreted phospholipase PLA2G2E contributes to regulation of T cell immune response against influenza virus infection.

The involvement of secreted phospholipase A2s in respiratory diseases, such as asthma and respiratory viral infections, is well-established. However, the specific role of secreted phospholipase A2 group IIE (PLA2G2E) during influenza virus infection remains unexplored. Here, we investigated the role of PLA2G2E during H1N1 influenza virus infection using a targeted mouse model lacking Pla2g2e gene (Pla2g2e-/-). Our findings demonstrated that Pla2g2e-/- mice had significantly lower survival rates and higher viral loads in lungs compared to wild-type mice following influenza virus infection. While Pla2g2e-/- mice displayed comparable innate and humoral immune responses to influenza virus challenge, the animals showed impaired influenza-specific cellular immunity and reduced T cell-mediated cytotoxicity. This indicates that PLA2G2E is involved in regulating specific T cell responses during influenza virus infection. Furthermore, transgenic mice expressing the human PLA2G2E gene exhibited resistance to influenza virus infection along with enhanced influenza-specific cellular immunity and T cell-mediated cytotoxicity. Pla2g2e deficiency resulted in perturbation of lipid mediators in the lung and T cells, potentially contributing to its impact on the anti-influenza immune response. Taken together, these findings suggest that targeting PLA2G2E could hold potential as a therapeutic strategy for managing influenza virus infections.IMPORTANCEThe influenza virus is a highly transmissible respiratory pathogen that continues to pose a significant public health concern. It effectively evades humoral immune protection conferred by vaccines and natural infection due to its continuous viral evolution through the genetic processes of antigenic drift and shift. Recognition of conserved non-mutable viral epitopes by T cells may provide broad immunity against influenza virus. In this study, we have demonstrated that phospholipase A2 group IIE (PLA2G2E) plays a crucial role in protecting against influenza virus infection through the regulation of T cell responses, while not affecting innate and humoral immune responses. Targeting PLA2G2E could therefore represent a potential therapeutic strategy for managing influenza virus infection.

High-throughput sequencing-based neutralization assay reveals how repeated vaccinations impact titers to recent human H1N1 influenza strains.

The high genetic diversity of influenza viruses means that traditional serological assays have too low throughput to measure serum antibody neutralization titers against all relevant strains. To overcome this challenge, we developed a sequencing-based neutralization assay that simultaneously measures titers against many viral strains using small serum volumes using a workflow similar to traditional neutralization assays. The key innovation is to incorporate unique nucleotide barcodes into the hemagglutinin (HA) genomic segment, and then pool viruses with numerous different barcoded HA variants and quantify the infectivity of all of them simultaneously using next-generation sequencing. With this approach, a single researcher performed the equivalent of 2,880 traditional neutralization assays (80 serum samples against 36 viral strains) in approximately 1 month. We applied the sequencing-based assay to quantify the impact of influenza vaccination on neutralization titers against recent human H1N1 strains for individuals who had or had not also received a vaccine in the previous year. We found that the viral strain specificities of the neutralizing antibodies elicited by vaccination vary among individuals and that vaccination induced a smaller increase in titers for individuals who had also received a vaccine the previous year-although the titers 6 months after vaccination were similar in individuals with and without the previous-year vaccination. We also identified a subset of individuals with low titers to a subclade of recent H1N1 even after vaccination. We provide an experimental protocol (dx.doi.org/10.17504/protocols.io.kqdg3xdmpg25/v1) and computational pipeline (https://github.com/jbloomlab/seqneut-pipeline) for the sequencing-based neutralization assays to facilitate the use of this method by others. IMPORTANCE: We describe a new approach that can rapidly measure how the antibodies in human serum inhibit infection by many different influenza strains. This new approach is useful for understanding how viral evolution affects antibody immunity. We apply the approach to study the effect of repeated influenza vaccination.

Headless hemagglutinin-containing influenza viral particles direct immune responses toward more conserved epitopes.

Seasonal influenza vaccines provide mostly strain-specific protection due to the elicitation of antibody responses focused on evolutionarily plastic antigenic sites in the hemagglutinin head domain. To direct the humoral response toward more conserved epitopes, we generated an influenza virus particle where the full-length hemagglutinin protein was replaced with a membrane-anchored, "headless" variant while retaining the normal complement of other viral structural proteins such as the neuraminidase as well as viral RNAs. We found that a single administration of a headless virus particle-based vaccine elicited high titers of antibodies that recognized more conserved epitopes on the major viral glycoproteins. Furthermore, the vaccine could elicit these responses even in the presence of pre-existing, hemagglutinin (HA) head-focused influenza immunity. Importantly, these antibody responses mediated protective, but non-neutralizing functions such as neuraminidase inhibition and antibody-dependent cellular cytotoxicity. Additionally, we show the vaccine can provide protection from homologous and heterologous challenges in mouse models of severe influenza without any measurable HA head-directed antibody responses. Thus, headless hemagglutinin containing viral particles may represent a tool to drive the types of antibody responses predicted to increase influenza vaccine breadth and durability.IMPORTANCECurrent seasonal influenza vaccines provide incomplete protection from disease. This is partially the result of the antibody response being directed toward parts of the virus that are tolerant of mutations. Redirecting the immune response to more conserved regions of the virus has been a central strategy of next-generation vaccine designs and approaches. Here, we develop and test a vaccine based on a modified influenza virus particle that expresses a partially deleted hemagglutinin protein along with the other viral structural proteins. We demonstrate this vaccine elicits antibodies that recognize the more conserved viral epitopes of the hemagglutinin stalk and neuraminidase protein to facilitate protection against influenza viruses despite a lack of classical viral neutralization activity.

Differential interferon responses to influenza A and B viruses in primary ferret respiratory epithelial cells.

Influenza B viruses (IBV) cocirculate with influenza A viruses (IAV) and cause periodic epidemics of disease, yet antibody and cellular responses following IBV infection are less well understood. Using the ferret model for antisera generation for influenza surveillance purposes, IAV resulted in robust antibody responses following infection, whereas IBV required an additional booster dose, over 85% of the time, to generate equivalent antibody titers. In this study, we utilized primary differentiated ferret nasal epithelial cells (FNECs) which were inoculated with IAV and IBV to study differences in innate immune responses which may result in differences in adaptive immune responses in the host. FNECs were inoculated with IAV (H1N1pdm09 and H3N2 subtypes) or IBV (B/Victoria and B/Yamagata lineages) and assessed for 72 h. Cells were analyzed for gene expression by quantitative real-time PCR, and apical and basolateral supernatants were assessed for virus kinetics and interferon (IFN), respectively. Similar virus kinetics were observed with IAV and IBV in FNECs. A comparison of gene expression and protein secretion profiles demonstrated that IBV-inoculated FNEC expressed delayed type-I/II IFN responses and reduced type-III IFN secretion compared to IAV-inoculated cells. Concurrently, gene expression of Thymic Stromal Lymphopoietin (TSLP), a type-III IFN-induced gene that enhances adaptive immune responses, was significantly downregulated in IBV-inoculated FNECs. Significant differences in other proinflammatory and adaptive genes were suppressed and delayed following IBV inoculation. Following IBV infection, ex vivo cell cultures derived from the ferret upper respiratory tract exhibited reduced and delayed innate responses which may contribute to reduced antibody responses in vivo.IMPORTANCEInfluenza B viruses (IBV) represent nearly one-quarter of all human influenza cases and are responsible for significant clinical and socioeconomic impacts but do not pose the same pandemic risks as influenza A viruses (IAV) and have thus received much less attention. IBV accounts for greater severity and deaths in children, and vaccine efficacy remains low. The ferret can be readily infected with human clinical isolates and demonstrates a similar course of disease and immune responses. IBV, however, generates lower antibodies in ferrets than IAV following the challenge. To determine whether differences in initial innate responses following infection may affect the development of robust adaptive immune responses, ferret respiratory tract cells were isolated, infected with IAV/IBV, and compared. Understanding the differences in the initial innate immune responses to IAV and IBV may be important in the development of more effective vaccines and interventions to generate more robust protective immune responses.

Characterization of T cell receptors reactive to HCRTNH2, pHA273-287, and NP17-31 in control and narcolepsy patients.

Narcolepsy type 1 (NT1), a disorder caused by hypocretin/orexin (HCRT) cell loss, is associated with human leukocyte antigen (HLA)-DQ0602 (98%) and T cell receptor (TCR) polymorphisms. Increased CD4+ T cell reactivity to HCRT, especially DQ0602-presented amidated C-terminal HCRT (HCRTNH2), has been reported, and homology with pHA273-287 flu antigens from pandemic 2009 H1N1, an established trigger of the disease, suggests molecular mimicry. In this work, we extended DQ0602 tetramer and dextramer data to 77 cases and 44 controls, replicating our prior finding and testing 709 TCRs in Jurkat 76 T cells for functional activation. We found that fewer TCRs isolated with HCRTNH2 (∼11%) versus pHA273-287 or NP17-31 antigens (∼50%) were activated by their ligand. Single-cell characterization did not reveal phenotype differences in influenza versus HCRTNH2-reactive T cells, and analysis of TCR CDR3αß sequences showed TCR clustering by responses to antigens but no cross-peptide class reactivity. Our results do not support the existence of molecular mimicry between HCRT and pHA273-287 or NP17-31.

Hemagglutination Inhibition Antibody Titers as Mediators of Influenza Vaccine Efficacy Against Symptomatic Influenza A(H1N1), A(H3N2), and B/Victoria Virus Infections.

BACKGROUND: The hemagglutination inhibition antibody (HAI) titer contributes only a part of vaccine-induced protection against influenza virus infections. Using causal mediation analysis, we quantified the proportion of vaccine efficacy mediated by postvaccination HAI titers. METHODS: We conducted causal mediation analyses using data from a randomized, active-comparator controlled, phase III, trial of an inactivated, split-virion seasonal quadrivalent influenza vaccine in children conducted from October 2010 to December 2011 in 8 countries. Vaccine efficacy was estimated using a weighted Cox proportional hazards model. Estimates were decomposed into the direct and indirect effects mediated by postvaccination HAI titers. RESULTS: The proportions of vaccine efficacy mediated by postvaccination HAI titers were estimated to be 22% (95% confidence interval, 18%--47%) for influenza A(H1N1), 20% (16%-39%) for influenza A(H3N2), and 37% (26%-85%) for influenza B/Victoria. CONCLUSIONS: HAI titers partially mediate influenza vaccine efficacy against influenza A(H1N1), A(H3N2), and B/Victoria. Our estimates were lower than in previous studies, possibly reflecting expected heterogeneity in antigenic similarity between vaccine and circulating viruses across seasons.