The Effect of Preexisting Immunity on Virus Detection and Immune Responses in a Phase II, Randomized Trial of a Russian-Backbone, Live, Attenuated Influenza Vaccine in Bangladeshi Children.

BACKGROUND: In a 2012 Phase II clinical trial, 300 Bangladeshi children aged 24 to 59 months with no prior influenza vaccine exposure were randomized to receive a single intranasally-administered dose of either trivalent, Russian-backbone, live, attenuated influenza vaccine (LAIV) or placebo. Protocol-defined analyses, presented in the companion manuscript, demonstrate decreased viral detection and immunogenicity for A/H1N1pdm09, relative to the A/H3N2 and B strains. This post hoc analysis of the trial data aims to investigate the LAIV strain differences by testing the hypothesis that preexisting humoral and mucosal immunity may influence viral recovery and immune responses after LAIV receipt. METHODS: We used logistic regressions to evaluate the relations between markers of preexisting immunity (ie, hemagglutination inhibition [HAI], microneutralization, and immunoglobulin G and immunoglobulin A (both serum and mucosal antibodies) and LAIV viral recovery in the week post-vaccination. We then tested for potential effect modification by baseline HAI titers (ie, <10 versus ≥10) and week 1 viral recovery on the LAIV-induced serum and mucosal immune responses, measured between days 0 and 21 post-vaccination. RESULTS: Higher levels of preexisting immunity to influenza A/H3N2 and B were strongly associated with strain-specific prevention of viral shedding upon LAIV receipt. While evidence of LAIV immunogenicity was observed for all 3 strains, the magnitudes of immune responses were most pronounced in children with no evidence of preexisting HAI and in those with detectable virus. CONCLUSIONS: The results provide evidence for a bidirectional association between viral replication and immunity, and underscore the importance of accounting for preexisting immunity when evaluating virologic and immunologic responses to LAIVs. CLINICAL TRIALS REGISTRATION: NCT01625689.

Immunogenicity and Viral Shedding of Russian-Backbone, Seasonal, Trivalent, Live, Attenuated Influenza Vaccine in a Phase II, Randomized, Placebo-Controlled Trial Among Preschool-Aged Children in Urban Bangladesh.

BACKGROUND: We evaluated a Russian-backbone, live, attenuated influenza vaccine (LAIV) for immunogenicity and viral shedding in a randomized, placebo-controlled trial among Bangladeshi children. METHODS: Healthy children received a single, intranasal dose of LAIV containing the 2011-2012 recommended formulation or placebo. Nasopharyngeal wash (NPW) specimens were collected on days 0, 2, 4, and 7. Reverse transcription polymerase chain reactions and sequencing identified the influenza virus (vaccine or wild-type). On days 0 and 21, blood specimens were collected to assess immunogenicity using hemagglutination inhibition, microneutralization, and immunoglobulin A (IgA) and G enzyme-linked immunosorbent assays (ELISAs); NPW specimens were also collected to assess mucosal immunogenicity using kinetic IgA ELISA. RESULTS: We enrolled 300 children aged 24 through 59 months in the immunogenicity and viral shedding analyses. Among children receiving LAIV, 45% and 67% shed A/H3N2 and B vaccine strains, respectively. No child shed A/H1N1 vaccine strain. There were significantly higher day 21 geometric mean titers (GMTs) for the LAIV, as compared to the placebo groups, in all immunoassays for A/H3N2 and B (log10 titer P < .0001; GMT Ratio >2.0). Among immunoassays for A/H1N1, only the mucosal IgA GMT was significantly higher than placebo at day 21 (log10 titer P = .0465). CONCLUSIONS: Children vaccinated with LAIV had serum and mucosal antibody responses to A/H3N2 and B, but only a mucosal IgA response to A/H1N1. Many children shed A/H3N2 and B vaccine strains, but none shed A/H1N1. More research is needed to determine the reason for decreased LAIV A/H1N1 immunogenicity and virus shedding. CLINICAL TRIALS REGISTRATION: NCT01625689.

Pathogenesis and transmission of avian influenza A (H7N9) virus in ferrets and mice.

On 29 March 2013, the Chinese Center for Disease Control and Prevention confirmed the first reported case of human infection with an avian influenza A(H7N9) virus. The recent human infections with H7N9 virus, totalling over 130 cases with 39 fatalities to date, have been characterized by severe pulmonary disease and acute respiratory distress syndrome (ARDS). This is concerning because H7 viruses have typically been associated with ocular disease in humans, rather than severe respiratory disease. This recent outbreak underscores the need to better understand the pathogenesis and transmission of these viruses in mammals. Here we assess the ability of A/Anhui/1/2013 and A/Shanghai/1/2013 (H7N9) viruses, isolated from fatal human cases, to cause disease in mice and ferrets and to transmit to naive animals. Both H7N9 viruses replicated to higher titre in human airway epithelial cells and in the respiratory tract of ferrets compared to a seasonal H3N2 virus. Moreover, the H7N9 viruses showed greater infectivity and lethality in mice compared to genetically related H7N9 and H9N2 viruses. The H7N9 viruses were readily transmitted to naive ferrets through direct contact but, unlike the seasonal H3N2 virus, did not transmit readily by respiratory droplets. The lack of efficient respiratory droplet transmission was corroborated by low receptor-binding specificity for human-like α2,6-linked sialosides. Our results indicate that H7N9 viruses have the capacity for efficient replication in mammals and human airway cells and highlight the need for continued public health surveillance of this emerging virus.

Kinetics of Serological Responses in Critically Ill Patients Hospitalized With 2009 Pandemic Influenza A(H1N1) Virus Infection in Canada, 2009-2011.

Background: The kinetics of the antibody response during severe influenza are not well documented. Methods: Critically ill patients infected with 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09), confirmed by reverse-transcription polymerase chain reaction analysis or seroconversion (defined as a ≥4-fold rise in titers), during 2009-2011 in Canada were prospectively studied. Antibody titers in serially collected sera were determined using hemagglutinin inhibition (HAI) and microneutralization assays. Average antibody curves were estimated using linear mixed-effects models and compared by patient outcome, age, and corticosteroid treatment. Results: Of 47 patients with A(H1N1)pdm09 virus infection (median age, 47 years), 59% had baseline HAI titers of <40, and 68% had baseline neutralizing titers of <40. Antibody titers rose quickly after symptom onset, and, by day 14, 83% of patients had HAI titers of ≥40, and 80% had neutralizing titers ≥40. Baseline HAI titers were significantly higher in patients who died compared with patients who survived; however, the antibody kinetics were similar by patient outcome and corticosteroid treatment. Geometric mean titers over time in older patients were lower than those in younger patients. Conclusions: Critically ill patients with influenza A(H1N1)pdm09 virus infection had strong HAI and neutralizing antibody responses during their illness. Antibody kinetics differed by age but were not associated with patient outcome.

Influence of Immune Priming and Egg Adaptation in the Vaccine on Antibody Responses to Circulating A(H1N1)pdm09 Viruses After Influenza Vaccination in Adults.

Background: Although ferret antisera used in influenza surveillance did not detect antigenic drift of A(H1N1)pdm09 viruses during the 2015-2016 season, low vaccine effectiveness was reported in adults. We investigated the immune basis of low responses to circulating A(H1N1)pdm09 viruses after vaccination. Methods: Prevaccination and postvaccination serum samples collected from >300 adults (aged 18-49 years) in 6 seasons (2010-2011 to 2015-2016) were analyzed using hemagglutination inhibition assays to evaluate the antibody responses to 13 A(H1N1) viruses circulated from 1977 to 2016. Microneutralization and serum adsorption assays were used to verify the 163K and 223R specificity of antibodies. Results: Individual antibody profiles to A(H1N1) viruses revealed 3 priming patterns: USSR/77, TW/86, or NC/99 priming. More than 20% of adults had reduced titers to cell-propagated circulating 6B.1 and 6B.2 A(H1N1)pdm09 viruses compared with the A/California/07/2009 vaccine virus X-179A. Significantly reduced antibody reactivity to circulating viruses bearing K163Q was observed only in the USSR/77-primed cohort, whereas significantly lower reactivity caused by egg-adapted Q223R change was detected across all 3 cohorts. Conclusion: Both 163K specificity driven by immune priming and 223R specificity from egg-adapted changes in the vaccine contributed to low responses to circulating A(H1N1)pdm09 viruses after vaccination. Our study highlights the need to incorporate human serology in influenza surveillance and vaccine strain selection.

Community-acquired pneumonia requiring hospitalization among U.S. children.

BACKGROUND: Incidence estimates of hospitalizations for community-acquired pneumonia among children in the United States that are based on prospective data collection are limited. Updated estimates of pneumonia that has been confirmed radiographically and with the use of current laboratory diagnostic tests are needed. METHODS: We conducted active population-based surveillance for community-acquired pneumonia requiring hospitalization among children younger than 18 years of age in three hospitals in Memphis, Nashville, and Salt Lake City. We excluded children with recent hospitalization or severe immunosuppression. Blood and respiratory specimens were systematically collected for pathogen detection with the use of multiple methods. Chest radiographs were reviewed independently by study radiologists. RESULTS: From January 2010 through June 2012, we enrolled 2638 of 3803 eligible children (69%), 2358 of whom (89%) had radiographic evidence of pneumonia. The median age of the children was 2 years (interquartile range, 1 to 6); 497 of 2358 children (21%) required intensive care, and 3 (<1%) died. Among 2222 children with radiographic evidence of pneumonia and with specimens available for bacterial and viral testing, a viral or bacterial pathogen was detected in 1802 (81%), one or more viruses in 1472 (66%), bacteria in 175 (8%), and both bacterial and viral pathogens in 155 (7%). The annual incidence of pneumonia was 15.7 cases per 10,000 children (95% confidence interval [CI], 14.9 to 16.5), with the highest rate among children younger than 2 years of age (62.2 cases per 10,000 children; 95% CI, 57.6 to 67.1). Respiratory syncytial virus was more common among children younger than 5 years of age than among older children (37% vs. 8%), as were adenovirus (15% vs. 3%) and human metapneumovirus (15% vs. 8%). Mycoplasma pneumoniae was more common among children 5 years of age or older than among younger children (19% vs. 3%). CONCLUSIONS: The burden of hospitalization for children with community-acquired pneumonia was highest among the very young, with respiratory viruses the most commonly detected causes of pneumonia. (Funded by the Influenza Division of the National Center for Immunization and Respiratory Diseases.).

Community-Acquired Pneumonia Requiring Hospitalization among U.S. Adults.

BACKGROUND: Community-acquired pneumonia is a leading infectious cause of hospitalization and death among U.S. adults. Incidence estimates of pneumonia confirmed radiographically and with the use of current laboratory diagnostic tests are needed. METHODS: We conducted active population-based surveillance for community-acquired pneumonia requiring hospitalization among adults 18 years of age or older in five hospitals in Chicago and Nashville. Patients with recent hospitalization or severe immunosuppression were excluded. Blood, urine, and respiratory specimens were systematically collected for culture, serologic testing, antigen detection, and molecular diagnostic testing. Study radiologists independently reviewed chest radiographs. We calculated population-based incidence rates of community-acquired pneumonia requiring hospitalization according to age and pathogen. RESULTS: From January 2010 through June 2012, we enrolled 2488 of 3634 eligible adults (68%). Among 2320 adults with radiographic evidence of pneumonia (93%), the median age of the patients was 57 years (interquartile range, 46 to 71); 498 patients (21%) required intensive care, and 52 (2%) died. Among 2259 patients who had radiographic evidence of pneumonia and specimens available for both bacterial and viral testing, a pathogen was detected in 853 (38%): one or more viruses in 530 (23%), bacteria in 247 (11%), bacterial and viral pathogens in 59 (3%), and a fungal or mycobacterial pathogen in 17 (1%). The most common pathogens were human rhinovirus (in 9% of patients), influenza virus (in 6%), and Streptococcus pneumoniae (in 5%). The annual incidence of pneumonia was 24.8 cases (95% confidence interval, 23.5 to 26.1) per 10,000 adults, with the highest rates among adults 65 to 79 years of age (63.0 cases per 10,000 adults) and those 80 years of age or older (164.3 cases per 10,000 adults). For each pathogen, the incidence increased with age. CONCLUSIONS: The incidence of community-acquired pneumonia requiring hospitalization was highest among the oldest adults. Despite current diagnostic tests, no pathogen was detected in the majority of patients. Respiratory viruses were detected more frequently than bacteria. (Funded by the Influenza Division of the National Center for Immunizations and Respiratory Diseases.).

Cross-Reactive Antibody Responses to Novel H5Nx Influenza Viruses Following Homologous and Heterologous Prime-Boost Vaccination with a Prepandemic Stockpiled A(H5N1) Vaccine in Humans.

Recently, novel highly pathogenic avian influenza H5Nx viruses (clade 2.3.4.4) caused outbreaks in US poultry. We evaluated the potential of a stockpiled A(H5N1) A/Anhui/1/2005 (clade 2.3.4) vaccine to elicit cross-reactive antibody responses to these emerging viruses. Sera from subjects who received 2 doses of MF59-adjuvanted A/Anhui/1/2005, or 1 dose of MF59-adjuvanted A/Anhui/1/2005 following priming with a clade 1 vaccine were characterized by microneutralization assays and modified hemagglutination inhibition (HI) assays. Only heterologous prime-boost vaccination induced modest cross-reactive HI antibody responses to H5Nx viruses. Heterologous prime-boost may provide a more effective vaccination strategy to broaden the antibody responses to emerging viruses.

Effect of Priming With Seasonal Influenza A(H3N2) Virus on the Prevalence of Cross-Reactive Hemagglutination-Inhibition Antibodies to Swine-Origin A(H3N2) Variants.

Background: Recent outbreaks of swine-origin influenza A(H3N2) variant (H3N2v) viruses have raised public health concerns. Previous studies indicated that older children and young adults had the highest levels of hemagglutination-inhibition (HI) antibodies to 2010-2011 H3N2v viruses. However, newly emerging 2013 H3N2v have acquired antigenic mutations in the hemagglutinin at amino acid position 145 (N145K/R). We estimated the levels of serologic cross-reactivity among humans primed with seasonal influenza A(H3N2) (sH3N2), using postinfection ferret antisera. We also explored age-related HI antibody responses to 2012-2013 H3N2v viruses. Methods: Human and ferret antisera were tested in HI assays against 1 representative 2012 H3N2v (145N) and 2 2013 H3N2v (145K/R) viruses, together with 9 sH3N2 viruses circulating since 1968. Results: Low levels of cross-reactivity between the H3N2v and sH3N2 viruses from the 1970s-1990s were observed using postinfection ferret antisera. The overall seroprevalence among the sH3N2-primed population against 2012-2013 H3N2v viruses was >50%, and age-related seroprevalence was observed. Seroprevalence was significantly higher to 2013 H3N2v than to 2012 H3N2v viruses among some children likely to have been primed with A/Sydney/5/97-like (145K) or A/Wuhan/359/95-like viruses (145K). Conclusions: A single substitution (N145K/R) was sufficient to affect seropositivity to H3N2v viruses in some individuals. Insight into age-related antibody responses to newly emerging H3N2v viruses is critical for risk assessment and pandemic preparedness.

A Pyrosequencing-Based Approach to High-Throughput Identification of Influenza A(H3N2) Virus Clades Harboring Antigenic Drift Variants.

The rapid evolution of influenza A(H3N2) viruses necessitates close monitoring of their antigenic properties so the emergence and spread of antigenic drift variants can be rapidly identified. Changes in hemagglutinin (HA) acquired by contemporary A(H3N2) viruses hinder antigenic characterization by traditional methods, thus complicating vaccine strain selection. Sequence-based approaches have been used to infer virus antigenicity; however, they are time consuming and mid-throughput. To facilitate virological surveillance and epidemiological studies, we developed and validated a pyrosequencing approach that enables identification of six HA clades of contemporary A(H3N2) viruses. The identification scheme of viruses of the H3 clades 3C.2, 3C.2a, 3C.2b, 3C.3, 3C.3a, and 3C.3b is based on the interrogation of five single nucleotide polymorphisms (SNPs) within three neighboring HA regions, namely 412 to 431, 465 to 481, and 559 to 571. Two bioinformatics tools, IdentiFire (Qiagen) and FireComb (developed in-house), were utilized to expedite pyrosequencing data analysis. The assay's analytical sensitivity was 10 focus forming units, and respiratory specimens with threshold cycle (CT) values of <34 typically produced good quality pyrograms. When applied to 120 A(H3N2) virus isolates and 27 respiratory specimens, the assay displayed 100% agreement with clades determined by HA sequencing coupled with phylogenetics. The multi-SNP analysis described here was readily adopted by another laboratory with pyrosequencing capabilities. The implementation of this approach enhanced the findings from virological surveillance and epidemiological studies between 2013 and 2016, which examined more than 3,000 A(H3N2) viruses.

Increase in Human Infections with Avian Influenza A(H7N9) Virus During the Fifth Epidemic - China, October 2016-February 2017.

During March 2013-February 24, 2017, annual epidemics of avian influenza A(H7N9) in China resulted in 1,258 avian influenza A(H7N9) virus infections in humans being reported to the World Health Organization (WHO) by the National Health and Family Planning Commission of China and other regional sources (1). During the first four epidemics, 88% of patients developed pneumonia, 68% were admitted to an intensive care unit, and 41% died (2). Candidate vaccine viruses (CVVs) were developed, and vaccine was manufactured based on representative viruses detected after the emergence of A(H7N9) virus in humans in 2013. During the ongoing fifth epidemic (beginning October 1, 2016),* 460 human infections with A(H7N9) virus have been reported, including 453 in mainland China, six associated with travel to mainland China from Hong Kong (four cases), Macao (one) and Taiwan (one), and one in an asymptomatic poultry worker in Macao (1). Although the clinical characteristics and risk factors for human infections do not appear to have changed (2,3), the reported human infections during the fifth epidemic represent a significant increase compared with the first four epidemics, which resulted in 135 (first epidemic), 320 (second), 226 (third), and 119 (fourth epidemic) human infections (2). Most human infections continue to result in severe respiratory illness and have been associated with poultry exposure. Although some limited human-to-human spread continues to be identified, no sustained human-to-human A(H7N9) transmission has been observed (2,3).

Update: Increase in Human Infections with Novel Asian Lineage Avian Influenza A(H7N9) Viruses During the Fifth Epidemic - China, October 1, 2016-August 7, 2017.

Among all influenza viruses assessed using CDC's Influenza Risk Assessment Tool (IRAT), the Asian lineage avian influenza A(H7N9) virus (Asian H7N9), first reported in China in March 2013,* is ranked as the influenza virus with the highest potential pandemic risk (1). During October 1, 2016-August 7, 2017, the National Health and Family Planning Commission of China; CDC, Taiwan; the Hong Kong Centre for Health Protection; and the Macao CDC reported 759 human infections with Asian H7N9 viruses, including 281 deaths, to the World Health Organization (WHO), making this the largest of the five epidemics of Asian H7N9 infections that have occurred since 2013 (Figure 1). This report summarizes new viral and epidemiologic features identified during the fifth epidemic of Asian H7N9 in China and summarizes ongoing measures to enhance pandemic preparedness. Infections in humans and poultry were reported from most areas of China, including provinces bordering other countries, indicating extensive, ongoing geographic spread. The risk to the general public is very low and most human infections were, and continue to be, associated with poultry exposure, especially at live bird markets in mainland China. Throughout the first four epidemics of Asian H7N9 infections, only low pathogenic avian influenza (LPAI) viruses were detected among human, poultry, and environmental specimens and samples. During the fifth epidemic, mutations were detected among some Asian H7N9 viruses, identifying the emergence of high pathogenic avian influenza (HPAI) viruses as well as viruses with reduced susceptibility to influenza antiviral medications recommended for treatment. Furthermore, the fifth-epidemic viruses diverged genetically into two separate lineages (Pearl River Delta lineage and Yangtze River Delta lineage), with Yangtze River Delta lineage viruses emerging as antigenically different compared with those from earlier epidemics. Because of its pandemic potential, candidate vaccine viruses (CVV) were produced in 2013 that have been used to make vaccines against Asian H7N9 viruses circulating at that time. CDC is working with partners to enhance surveillance for Asian H7N9 viruses in humans and poultry, to improve laboratory capability to detect and characterize H7N9 viruses, and to develop, test and distribute new CVV that could be used for vaccine production if a vaccine is needed.

Seasonal Influenza Vaccination of Children Induces Humoral and Cell-Mediated Immunity Beyond the Current Season: Cross-reactivity With Past and Future Strains.

BACKGROUND: Influenza viruses gradually accumulate point mutations, reducing the effectiveness of prior immune protection. METHODS: Children aged 9-14 years received 2010-2011 trivalent inactivated influenza vaccine (TIV). Vaccination history, hemagglutination-inhibition (HI) titers, and cell-mediated immune responses were assessed to investigate the cross-reactivity with past and future influenza virus strains. RESULTS: 2010-2011 TIV induced significant T-cell responses and HI titers of ≥160, with a fold-rise of ≥4 and titers of ≥100 maintained for >7 months in the majority of children. Pre-existing memory B cells in these children differentiated quickly to antibody-secreting cells to the new vaccine antigens. Children vaccinated in the previous year maintained high HI titers well into 2010, demonstrating elevated HI titers against A/Perth/16/2009, the future (in 2010-2011) H3N2 component. Prior vaccination enhanced CD8+ T-cell responses to A/Perth/16/2009. Children vaccinated with the prior 2009-2010 seasonal vaccine also demonstrated higher preexisting levels of interferon γ-secreting CD4+CD69+ T cells to 2009 pandemic influenza A(H1N1). Children previously vaccinated with 2009-2010 seasonal influenza vaccine also showed greater expansion of tumor necrosis factor α-secreting CD8+CD69+ T cells to 2009 pandemic influenza A(H1N1) upon vaccination in the 2010-2011 season than those who were not previously vaccinated. CONCLUSIONS: Seasonal influenza viruses continuously drift, which allows them to circumvent protective immunity, but conserved epitopes provide immunological cross-reactivity in children through either vaccination directly or through prime/boost in the prior influenza season.

Randomized Controlled Trial to Compare Immunogenicity of Standard-Dose Intramuscular Versus Intradermal Trivalent Inactivated Influenza Vaccine in HIV-Infected Men Who Have Sex With Men in Bangkok, Thailand.

BACKGROUND: Individuals infected with human immunodeficiency virus (HIV) are at increased risk for severe influenza, yet immune responses to standard-dose intramuscular (IM) influenza vaccine are suboptimal in this population. Intradermal (ID) delivery of influenza vaccine might improve immune response through enhanced stimulation of dendritic cells. METHODS: We conducted a randomized, double-blind, controlled trial to compare the immunogenicity of off-label standard-dose (15 µg) ID vs standard-dose (15 µg) IM inactive influenza vaccine in HIV-infected men in Bangkok, Thailand. The primary study outcome was seroconversion (minimum titer of 1:40 and ≥4-fold rise in antibody titer) at 1 month postvaccination based on serum hemagglutination inhibition antibody titers against each vaccine strain. Adverse events (AEs) in the 7 days following vaccination were also assessed. RESULTS: We enrolled 400 HIV-infected participants; 200 were randomly assigned to receive IM and 200 ID vaccine. Vaccine arms were well-balanced with respect to age, CD4 cell count, HIV RNA load, and antiretroviral treatment. Percentage of seroconversion to all (ID 14% vs IM 15%; P = .8) or at least 1 (ID 69% vs IM 68%; P = .7) of the 3 vaccine strains did not differ significantly between ID vs IM vaccine recipients. A higher proportion of participants who received ID vaccine had mild injection-site AEs compared with participants who received IM vaccine (77% vs 27%). CONCLUSIONS: There were no significant differences in the immunogenicity of standard-dose ID vs IM influenza vaccine in this HIV-infected population in Thailand. Additional strategies to enhance immune responses to influenza vaccine among HIV-infected persons are needed. CLINICAL TRIALS REGISTRATION: NCT01538940.

NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection.

The NLR protein, NLRC5 is an important regulator of MHC class I gene expression, however, the role of NLRC5 in other innate immune responses is less well defined. In the present study, we report that NLRC5 binds RIG-I and that this interaction is critical for robust antiviral responses against influenza virus. Overexpression of NLRC5 in the human lung epithelial cell line, A549, and normal human bronchial epithelial cells resulted in impaired replication of influenza virus A/Puerto Rico/8/34 virus (PR8) and enhanced IFN-ß expression. Influenza virus leads to induction of IFN-ß that drives RIG-I and NLRC5 expression in host cells. Our results suggest that NLRC5 extends and stabilizes influenza virus induced RIG-I expression and delays expression of the viral inhibitor protein NS1. We show that NS1 binds to NLRC5 to suppress its function. Interaction domain mapping revealed that NLRC5 interacts with RIG-I via its N-terminal death domain and that NLRC5 enhanced antiviral activity in an leucine-rich repeat domain independent manner. Taken together, our findings identify a novel role for NLRC5 in RIG-I-mediated antiviral host responses against influenza virus infection, distinguished from the role of NLRC5 in MHC class I gene regulation.

A(H7N9) virus results in early induction of proinflammatory cytokine responses in both human lung epithelial and endothelial cells and shows increased human adaptation compared with avian H5N1 virus.

UNLABELLED: Similar to H5N1 viruses, A(H7N9) influenza viruses have been associated with severe respiratory disease and fatal outcomes in humans. While high viral load, hypercytokinemia, and pulmonary endothelial cell involvement are known to be hallmarks of H5N1 virus infection, the pathogenic mechanism of the A(H7N9) virus in humans is largely unknown. In this study, we assessed the ability of A(H7N9) virus to infect, replicate, and elicit innate immune responses in both human bronchial epithelial cells and pulmonary microvascular endothelial cells, compared with the abilities of seasonal H3N2, avian H7N9, and H5N1 viruses. In epithelial cells, A(H7N9) virus replicated efficiently but did not elicit robust induction of cytokines like that observed for H5N1 virus. In pulmonary endothelial cells, A(H7N9) virus efficiently initiated infection; however, no released infectious virus was detected. The magnitudes of induction of host cytokine responses were comparable between A(H7N9) and H5N1 virus infection. Additionally, we utilized differentiated human primary bronchial and tracheal epithelial cells to investigate cellular tropism using transmission electron microscopy and the impact of temperature on virus replication. Interestingly, A(H7N9) virus budded from the surfaces of both ciliated and mucin-secretory cells. Furthermore, A(H7N9) virus replicated to a significantly higher titer at 37 °C than at 33 °C, with improved replication capacity at 33 °C compared to that of H5N1 virus. These findings suggest that a high viral load from lung epithelial cells coupled with induction of host responses in endothelial cells may contribute to the severe pulmonary disease observed following H7N9 virus infection. Improved adaptation of A(H7N9) virus to human upper airway poses an important threat to public health. IMPORTANCE: A(H7N9) influenza viruses have caused over 450 documented human infections with a 30% fatality rate since early 2013. However, these novel viruses lack many molecular determinants previously identified with mammalian pathogenicity, necessitating a closer examination of how these viruses elicit host responses which could be detrimental. This study provides greater insight into the interaction of this virus with host lung epithelial cells and endothelial cells, which results in high viral load, epithelial cell death, and elevated immune response in the lungs, revealing the mechanism of pathogenesis and disease development among A(H7N9)-infected patients. In particular, we characterized the involvement of pulmonary endothelial cells, a cell type in the human lung accessible to influenza virus following damage of the epithelial monolayer, and its potential role in the development of severe pneumonia caused by A(H7N9) infection in humans.

Emergence of Highly Pathogenic Avian Influenza A(H5N1) Virus PB1-F2 Variants and Their Virulence in BALB/c Mice.

UNLABELLED: Influenza A viruses (IAVs) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 proteins of mammalian IAVs frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAVs usually express a full-length 90-amino-acid PB1-F2. However, in contrast to other avian IAVs, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1,000-fold more virulent for BALB/c mice than a closely related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length PB1-F2, truncated (24-amino-acid) PB1-F2, and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full-length and C-terminal PB1-F2 mutants colocalized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. IMPORTANCE: Although most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses.

An oil-in-water nanoemulsion enhances immunogenicity of H5N1 vaccine in mice.

To enhance the immunogenicity of the Influenza H5N1 vaccine, we developed an oil-in-water nanoemulsion (NE) adjuvant. NE displayed good temperature stability and maintained particle size. More importantly, it significantly enhanced IL-6 and MCP-1 production to recruit innate cells, including neutrophils, monocytes/macrophages and dendritic cells to the local environment. Furthermore, NE enhanced dendritic cell function to induce robust antigen-specific T and B cell immune responses. NE-adjuvanted H5N1 vaccine not only elicited significantly higher and long-lasting antibody responses, but also conferred enhanced protection against homologous clade 1 as well as heterologous clade 2 H5N1 virus challenge in young as well as in aged mice. The pre-existing immunity to seasonal influenza did not affect the immunogenicity of NE-adjuvanted H5N1 vaccine.

Antibody to Influenza Virus Neuraminidase: An Independent Correlate of Protection.

BACKGROUND: Laboratory correlates of influenza vaccine protection can best be identified by examining people who are infected despite vaccination. While the importance of antibody to viral hemagglutinin (HA) has long been recognized, the level of protection contributed independently by antibody to viral neuraminidase (NA) has not been determined. METHODS: Sera from a controlled trial of the efficacies of inactivated influenza vaccine (IIV) and live attenuated influenza vaccine (LAIV) were tested by hemagglutination inhibition (HAI) assay, microneutralization (MN) assay, and a newly standardized lectin-based neuraminidase inhibition (NAI) assay. RESULTS: The NAI assay detected a vaccine response in 37% of IIV recipients, compared with 77% and 67% of participants in whom responses were detected by the HAI and MN assays, respectively. For LAIV recipients, the NAI, HAI, and MN assays detected responses in 6%, 21%, and 17%, respectively. In IIV recipients, as NAI assay titers rose, the frequency of infection fell, similar to patterns seen with HAI and MN assays. HAI and MN assay titers were highly correlated, but NAI assay titers exhibited less of a correlation. Analyses suggested an independent role for NAI antibody in protection, which was similar in the IIV, LAIV, and placebo groups. CONCLUSIONS: While NAI antibody is not produced to a large extent in response to current IIV, it appears to have an independent role in protection. As new influenza vaccines are developed, NA content should be considered. CLINICAL TRIALS REGISTRATION: NCT00538512.

A Newly Emerged Swine-Origin Influenza A(H3N2) Variant Dampens Host Antiviral Immunity but Induces Potent Inflammasome Activation.

We compared the innate immune response to a newly emerged swine-origin influenza A(H3N2) variant containing the M gene from 2009 pandemic influenza A(H1N1), termed "A(H3N2)vpM," to the immune responses to the 2010 swine-origin influenza A(H3N2) variant and seasonal influenza A(H3N2). Our results demonstrated that A(H3N2)vpM-induced myeloid dendritic cells secreted significantly lower levels of type I interferon (IFN) but produced significantly higher levels of proinflammatory cytokines and induced potent inflammasome activation. The reduction in antiviral immunity with increased inflammatory responses upon A(H3N2)vpM infection suggest that these viruses have the potential for increased disease severity in susceptible hosts.