Development and use of lentiviral vectors pseudotyped with influenza B haemagglutinins: application to vaccine immunogenicity, mAb potency and sero-surveillance studies

Influenza B viruses cause respiratory disease epidemics in human populations and are included in seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However, the haemagglutination inhibition assay, which represents the gold standard for assessing the immunogenicity of influenza vaccines, has been shown to be relatively insensitive for the detection of antibodies against influenza B viruses. Furthermore, this assay, and the serial radial haemolysis assay are not able to detect stalk-directed cross-reactive antibodies. For these reasons there is a need to develop new assays that can overcome these limitations. The use of replication-defective viruses, such as lentiviral vectors pseudotyped with influenza A haemagglutinins, in microneutralization assays is a safe and sensitive alternative to study antibody responses elicited by natural infection or vaccination. We have produced Influenza B haemagglutinin-pseudotypes using plasmid-directed transfection. To activate influenza B haemagglutinin, we have explored the use of proteases by adding relevant encoding plasmids to the transfection mixture. When tested for their ability to transduce target cells, the newly produced influenza B pseudotypes exhibit tropism for different cell lines. Subsequently the pseudotypes were evaluated as surrogate antigens in microneutralization assays using reference sera, monoclonal antibodies, human sera collected during a vaccine immunogenicity study and surveillance sera from seals. The influenza B pseudotype virus neutralization assay was found to effectively detect neutralizing and cross-reactive responses despite lack of significant correlation with the haemagglutinin inhibition assay

Ready, Set, Fuse! The Coronavirus Spike Protein and Acquisition of Fusion Competence

Coronavirus-cell entry programs involve virus-cell membrane fusions mediated by viral spike (S) proteins. Coronavirus S proteins acquire membrane fusion competence by receptor interactions, proteolysis, and acidification in endosomes. This review describes our current understanding of the S proteins, their interactions with and their responses to these entry triggers. We focus on receptors and proteases in prompting entry and highlight the type II transmembrane serine proteases (TTSPs) known to activate several virus fusion proteins. These and other proteases are essential cofactors permitting coronavirus infection, conceivably being in proximity to cell-surface receptors and thus poised to split entering spike proteins into the fragments that refold to mediate membrane fusion. The review concludes by noting how understanding of coronavirus entry informs antiviral therapies

MicroRNA Regulation of Human Protease Genes Essential for Influenza Virus Replication

Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies

Innate Immune Response of Human Alveolar Macrophages during Influenza A Infection

Alveolar macrophages (AM) are one of the key cell types for initiating inflammatory and immune responses to influenza virus in the lung. However, the genome-wide changes in response to influenza infection in AM have not been defined. We performed gene profiling of human AM in response to H1N1 influenza A virus PR/8 using Affymetrix HG-U133 Plus 2.0 chips and verified the changes at both mRNA and protein levels by real-time RT-PCR and ELISA. We confirmed the response with a contemporary H3N2 influenza virus A/New York/238/2005 (NY/238). To understand the local cellular response, we also evaluated the impact of paracrine factors on virus-induced chemokine and cytokine secretion. In addition, we investigated the changes in the expression of macrophage receptors and uptake of pathogens after PR/8 infection. Although macrophages fail to release a large amount of infectious virus, we observed a robust induction of type I and type III interferons and several cytokines and chemokines following influenza infection. CXCL9, 10, and 11 were the most highly induced chemokines by influenza infection. UV-inactivation abolished virus-induced cytokine and chemokine response, with the exception of CXCL10. The contemporary influenza virus NY/238 infection of AM induced a similar response as PR/8. Inhibition of TNF and/or IL-1β activity significantly decreased the secretion of the proinflammatory chemokines CCL5 and CXCL8 by over 50%. PR/8 infection also significantly decreased mRNA levels of macrophage receptors including C-type lectin domain family 7 member A (CLEC7A), macrophage scavenger receptor 1 (MSR1), and CD36, and reduced uptake of zymosan. In conclusion, influenza infection induced an extensive proinflammatory response in human AM. Targeting local components of innate immune response might provide a strategy for controlling influenza A infection-induced proinflammatory response in vivo