In this core expression-cloning technologies will be utilized to generate a collection of mAbs that will be characterized in various contexts by the three projects. Further, arrays of hundreds or thousands of antibodies will be printed to protein microarrays for rapid screening of mAb or poly-clonal (serum) antibody epitope targeting. We will clone influenza-specific antibodies from plasmablasts activated after vaccination of healthy volunteers. As we have shown 50 to 70% of influenza-vaccine induced plasmablasts are influenza specific. Thus this effort will generate a large library of anti-HA and anti-NA human mAbs to the three or four vaccine strains. In order to isolate a larger library of NA-reactive antibodies we will use fluorescently labeled neuraminidase (NA) protein as bait to identify and sort anti-NA memory B cells from human PBMCs from patients infected with influenza. Finally, we have a substantial collection of human mAbs both cloned in our laboratory and canonical antibodies from the literature. These antibodies bind to all recent influenza vaccine strains and to various different proteins (HA, NA, NP, M) and epitopes in the HA-stalk and HA-head regions. Using microarrays of these printed antibodies we can readily determine the epitopes shared between recombinant HAs and whole virions. Further, using what we term "epitope-shadowing" we can screen for global changes in the epitope specificity of serum samples by competition binding assays. This powerful tool will be further developed as a platform to use for similar experiments and to identify serum-specificity signatures correlated with protection (biomarker applications), to bin the "paratope" of protective and non protective epitopes typically targeted, and to use as a powerful tool to quickly map epitopes targeted by new antibodies or sera. The antibody core services will be used by the various projects as follows: Project 1 will characterize conserved epitopes and mechanisms of neutralization from antibodies targeting both HA and NA proteins and will use the antibody arrays to screen for epitope specificities. Project 2 will use the antibody core to generate antibodies to determine how anti-influenza responses become skewed in particular cohorts and to scrutinize the specificity of novel memory B cell populations. The antibody microarray will be used to evaluate epitopes targeted by human subjects or by B cell subsets. Project 3 will use the antibodies to Fc FcR engagement to the mechanisms of antibody-mediated protection from influenza.
With the discovery of many broadly-protective epitopes allowing neutralization of multiple influenza strains the generation of a broadly-protective one-shot influenza vaccine has become an almost tenable goal in recent years. Each of the projects in this program centers on aspects of antibody responses and the antibody core will provide antibodies and antibody microarrays to allow high throughput screening of antibody activities.
|Ho, Irvin Y; Bunker, Jeffrey J; Erickson, Steven A et al. (2016) Refined protocol for generating monoclonal antibodies from single human and murine B cells. J Immunol Methods 438:67-70|
|Chen, Chi-Jene; Ermler, Megan E; Tan, Gene S et al. (2016) Influenza A Viruses Expressing Intra- or Intergroup Chimeric Hemagglutinins. J Virol 90:3789-93|
|Krammer, Florian (2016) Novel universal influenza virus vaccine approaches. Curr Opin Virol 17:95-103|
|Nachbagauer, Raffael; Choi, Angela; Izikson, Ruvim et al. (2016) Age Dependence and Isotype Specificity of Influenza Virus Hemagglutinin Stalk-Reactive Antibodies in Humans. MBio 7:e01996-15|
|Tan, Gene S; Leon, Paul E; Albrecht, Randy A et al. (2016) Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection. PLoS Pathog 12:e1005578|
|He, Wenqian; Tan, Gene S; Mullarkey, Caitlin E et al. (2016) Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus. Proc Natl Acad Sci U S A 113:11931-11936|
|Neu, Karlynn E; Henry Dunand, Carole J; Wilson, Patrick C (2016) Heads, stalks and everything else: how can antibodies eradicate influenza as a human disease? Curr Opin Immunol 42:48-55|
|DiLillo, David J; Palese, Peter; Wilson, Patrick C et al. (2016) Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection. J Clin Invest 126:605-10|
|Henry Dunand, Carole J; Leon, Paul E; Huang, Min et al. (2016) Both Neutralizing and Non-Neutralizing Human H7N9 Influenza Vaccine-Induced Monoclonal Antibodies Confer Protection. Cell Host Microbe 19:800-13|
|Hamilton, Jennifer R; Sachs, David; Lim, Jean K et al. (2016) Club cells surviving influenza A virus infection induce temporary nonspecific antiviral immunity. Proc Natl Acad Sci U S A 113:3861-6|
Showing the most recent 10 out of 39 publications