The ectodomain of influenza matrix protein 2 (M2e) may be an ideal candidate in the development of influenza universal vaccine due to its highly conserved property among different subtypes/strains of influenza virus. M2e based vaccines have been extensively studied and potent cross-subtype/strain protections have been reported. However, more and more M2e mutants of influenza virus have been identified in recent years. It is still unclear whether M2e based vaccines are effective against these M2e mutants of influenza virus. This study first evaluated cross-protection of an M2e tetrameric peptide vaccine based on H5N1 virus strain A/Vietnam/1194/04 (VN/1194-M2e) against lethal challenges of M2e mutants of H5N1 virus strain A/Hong Kong/156/97 (HK/156) and a novel H7N9 virus, because there are 3 or 5 amino acid differences between VN/1194-M2e and HK/156-M2e or VN/1194-M2e and H7N9-M2e. The results showed that the vaccination of VN/1194-M2e did not induce high level of cross-reactive antibodies against HK/156-M2e and just provided poor cross-protection against lethal challenge of HK/156 virus. In contrast, VN/1194-M2e vaccination induced high level of cross-reactive antibodies against H7N9-M2e. Consistently, the vaccination provided good cross-protection against lethal challenge of H7N9 virus. These results strongly suggested that some mutations in M2e, such as mutations at positions 10, 14 and 16 which found in HK/156 M2e, might affect the M2e vaccine efficacy, but some others, such as five mutations found in H7N9-M2e, might not be critical for the M2e immunogenicity. This study then investigated the relationship between the M2e immunogenicity and amino acid mutations of the M2e. Beside VN/1194-M2e (P0), we synthesized additional 10 M2e mutant peptides which contain different single or multiple mutations. The 3D structures of these M2e peptides were predicted and analyzed. The prediction results showed that group 1 peptides (P0, P10, P14, P16, P18, P20 and P18-20) exhibited either irregular structures or loose hairpin structures which might associate with well exposure of antigenic epitope, whereas group 2 peptides (P10-14, P10-16, P14-16 and P10-14-16) formed tight hairpin structures in which antigenic epitope might bury inside their own secondary structure. Vaccination efficacies of these M2e peptides were evaluated in mice for antibody responses and cross-protection against lethal challenge of VN/1194 and HK/156 viruses. Our results showed that vaccinations of group 1 peptides induced high levels of cross-reactive antibodies against VN/1194-M2e and good cross-protection against lethal challenge of VN/1194 virus. However, vaccinations of group 2 peptides vaccinations induced significantly lower VN/1194-M2e antibody responses and poor cross-protection against lethal challenge of VN/119 virus. Furthermore, both group 1 and group 2 peptides could just induce low levels of cross-reactive antibodies against HK/156-M2e and poor protection against lethal challenge of HK/156 virus. Although H5N1-M2e tetrameric peptide has been previously shown to protect mice from lethal challenges by different subtypes/strains of influenza virus, this study has shown that certain amino acid variations in M2e could weaken M2e immunogenicity but some others might not. The different secondary structures of M2es may probably associate with their immunogenicity. Our findings have provided valuable information for the development of M2e based universal vaccines. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Human infections with influenza A virus (IAV) exhibit mild to severe clinical outcomes as a result of differential virus-host interactions. C-type lectin receptors (CLRs) are pattern recognition receptors that may sense carbohydrates, proteins, or lipids derived from infected hosts or the invading microbes including bacteria, viruses, fungi, or parasites. CLR-viral interaction may lead to increased viral entry and spread; furthermore, their interactions have been reported to trigger downstream signaling that further modulates host’s innate immune responses through the induction of pro-inflammatory cytokines. To date, DC-SIGN and DC-SIGNR have been shown to mediate IAV entry; however, the potential interactions between other human transmembrane CLRs with IAV have not yet been systematically investigated. We utilized lentiviral-based pseudoparticles expressing influenza hemagglutinin (HA) to examine the binding potential between HA and a panel of human CLRs expressed in soluble form. CLEC5A was identified as a potential interacting target with the HA proteins derived from a highly pathogenic avian H5N1 virus A/VN/1203/04 (VN1203) or a human seasonal H1N1 virus A/HK/54/98 (HK5498), albeit at different binding intensity. Applying siRNA gene silencing, we confirmed that CLEC5A did not enhance influenza entry in human monocytic U937 cells that constitutively express CLEC5A or in the lentiviral-transduced stable CHO and CHO-Lec2 cells that overly expressed CLEC5A. To investigate downstream signaling upon engagement of CLEC5A to influenza virus, M-CSF or GM-CSF differentiated human macrophages with high expression levels of CLEC5A and DAP12, a known adaptor protein for CLEC5A upon phosphorylation to initiate signal transduction, was subjected to CLEC5A siRNA gene silencing followed by infection with recombinant A/PR/8/34 virus expressing HA and NA derived from either VN1203 (H5N1) or HK5498 (H1N1) viruses. RG-PR8xVN1203HA,NA (H5N1) exhibited a higher infectivity and induced higher levels of pro-inflammatory cytokines (TNF-( and IFN-α) and chemokines (IP-10, MCP-1, MIG and MIP-1α) secretion in M-CSF or GM-CSF differentiated macrophages while compared to that of the RG-PR8xHK5498HA,NA (H1N1) virus. Knocking-down CLEC5A in macrophages led to a universal reduction of cytokines and chemokines secretion after infection with either the RG-PR8xVN1203HA,NA, RG-PR8xHK5498HA,NA, RG-A/VN/1203/04 (H5N1) or A/Shanghai/2/2014 (H7N9) viruses, suggesting that CLEC5A plays a role as cytokine and chemokine amplifier after influenza infection. Since DAP12 phosphorylation is known to activate downstream signaling via Spleen tyrosine kinase (Syk), pre-incubation of M-CSF macrophages with a Syk inhibitor (Bay 61-3606) also lead to a significant reduction of TNF-α and IP-10 in infected macrophages. A higher mortality was observed in CLEC5A-/- mice while compared to the wild-type C57BL/6 mice after challenged with a lethal dose of RG-A/VN/1203/04 (H5N1) influenza virus suggesting that CLEC5A as a host innate response amplifier play a protective role upon influenza infection. In conclusion, we have identified CLEC5A as a novel host factor for influenza pathogenesis by modulating host innate inflammatory response. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

No description available.

No description available.

No description available.

No description available.

Background: Influenza is a highly contagious but preventable acute respiratory illness associated with high morbidity. Seasonal influenza affects approximately 20% to 40% of children and adolescents. Annual influenza vaccination is an effective approach to prevent illness but recent studies suggests that adolescents are underutilizing important preventive health services and that influenza vaccination coverage in high risk adolescents is also suboptimal. The purpose of this study was to examine the association between household reported health status and socio-demographic characteristics of U.S. adolescents who reported receiving an influenza vaccination. Methods: Data from the 2008 National Immunization Survey were assessed examining various demographic and socioeconomic characteristics, as well as reported health status of non-institutionalized adolescents in the U.S. The sample was limited adolescents aged 13-17. Odds ratios were calculated and multivariate logistic regression was conducted. P-values of < 0.05 and 95% confidence intervals were used to determine statistical significance. Results: There were 29063 total observations with 18.9% reporting receiving the influenza vaccine. The results of this study indicate that sex, race and ethnicity, poverty status, health insurance status, asthma status, having an underlying health condition, missed school days due to illness or injury, and maternal age are associated with getting immunized against influenza. As one might expect those who reported having health insurance, having asthma, and having an underlying health condition had higher likelihood of vaccine. Interestingly, non-Hispanic other race and multi-race teens in the study were the most likely to receive the influenza vaccine compared with non-Hispanic white teens. Conclusions: This study further examines the impact of socio-demographic disparities and health status on influenza vaccination coverage. Although the current influenza vaccine recommendations now include all individuals ages 6 months and older, it should still be important to recognize disparities and inequalities which contribute to non-vaccination or under-vaccination. Improved understanding of demographic and socioeconomic characteristics, as well as existing underlying health conditions, will facilitate the path to improving interventions, vaccination rates, and subsequent reduction in the burden of this preventable disease.

No description available.

No description available.

The interferon (IFN) system is integral to antiviral innate immunity in vertebrate hosts. Inside a cell, viral pathogen associated molecular patterns (PAMPs) trigger the IFN response, comprised of IFN induction and an IFN-induced antiviral state. However, viruses have evolved strategies to counteract the IFN system. The E3 protein of vaccinia virus (VV), encoded by the E3L gene, impedes cytokine expression and suppresses the activation and function of antiviral proteins. Deletion of the E3L gene (VVΔE3L) produces an IFN sensitive mutant virus that is replication defective in most human cell lines. Due to the limited human cell lines available to support VVΔE3L replication, the capacity of E3 inhibition of human IFN-induced antiviral activities is not well defined. In this study, VVΔE3L was generated and characterized to facilitate the study of other viral IFN antagonists at modulating human IFN-induced antiviral responses. A human liver carcinoma cell line, Huh7, was found to support VVΔE3L replication. A comprehensive analysis of VVΔE3L IFN sensitivity revealed E3 inhibits all human type I and type II IFN-induced antiviral activities by modulation of the protein kinase R (PKR) pathway. Influenza non-structural protein 1 (NS1) is well-known to mediate the suppression of IFN induction and IFN action in influenza virus infections. However, the IFN antagonizing potential of influenza NS1 may be virus subtype and/or isolate specific. VVΔE3L was next applied as an expression vector to study influenza NS1 function in modulating IFN-induced antiviral activities and IFN induction in human cells. Recombinant viruses were generated to express influenza NS1 (from avian H5N1 and pandemic viruses 1918 pH1N1, 1968 pH3N2, and 2009 pH1N1) in replacement of E3. It was found that influenza NS1 inhibits human IFN-induced antiviral activity in a subtype and isolate specific manner. Moreover, influenza NS1 differentially regulates human IFN expression in a virus isolate-dependent manner. Altogether, this work highlights the potential of VVΔE3L as an excellent virus model system to study viral proteins modulating IFN expression and IFN-induced antiviral activities in human cells.