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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program--Cooperative Agreements (U19)
Project #
1U19AI109946-01
Application #
8667702
Study Section
Special Emphasis Panel (ZAI1-ZL-I (J1))
Project Start
Project End
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
1
Fiscal Year
2014
Total Cost
$242,911
Indirect Cost
$41,741
Name
Icahn School of Medicine at Mount Sinai
Department
Type
DUNS #
078861598
City
New York
State
NY
Country
United States
Zip Code
10029
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Ellebedy, Ali H; Krammer, Florian; Li, Gui-Mei et al. (2014) Induction of broadly cross-reactive antibody responses to the influenza HA stem region following H5N1 vaccination in humans. Proc Natl Acad Sci U S A 111:13133-8
Tan, Gene S; Lee, Peter S; Hoffman, Ryan M B et al. (2014) Characterization of a broadly neutralizing monoclonal antibody that targets the fusion domain of group 2 influenza A virus hemagglutinin. J Virol 88:13580-92
Nachbagauer, Raffael; Wohlbold, Teddy John; Hirsh, Ariana et al. (2014) Induction of broadly reactive anti-hemagglutinin stalk antibodies by an H5N1 vaccine in humans. J Virol 88:13260-8