Influenza Virus

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Fig. 1. Fine epitope mapping of anti-H5 2A-scFvFc antibody. A. Flow cytometry profiles for immunoreactivity of single-clone HA1-M mutants displayed on the surface of yeast. Single point mutations that abolish yeast surface binding of anti-H5 2AscFvFc were analyzed and mapped to distinct regions of HA1. B. Schematic representation of the epitopes recognized by anti-H5 2AFc to HA1 on the yeast surface. Amino acid positions are designated in H5 numbering. A linear epitopes (aa 206-211) recognized by 2AFc is colored in red. The regions encompassing the receptor-binding domain are colored in cyan.

Specific Aim 3. Define the neutralization escape pathways for each neutralizing Mab. Are there common or unique pathways for each Mab. Is it a stochastic process or does it follow a defined, reproducible pathway due to genetic constraints.

Aim 3 studies and progress:

We have aimed to generate escape mutants under the selection of our highly potent neutralizing antibody F10 which has been extensively characterized for structural insights into the mechanisms of epitope-specific neutralization. This F10 antibody is particularly valuable for the immune-driven viral evolution studies as this antibody targets highly conserved pocket in the stem region shared with the diverse influenza subtyupes and function critical for viral fusion. Therefore, characterizing permitted evolutionary routes of the virus over the course of F10-type immune selection may serve as templates for the design of universal influenza vaccine and treatment strategies against all types of influenza viruses including those emergent pandemic strains. Selection of VN/04 (H5N1) or A/PR/8 virus escape mutants with F10 antibody was performed in MDCK cells under conditio...

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...nd effects on clinical response and outcome of infections with immune-driven escape mutants. Defining neutralization escape pathways for potent neutralizing human monoclonal antibodies may further serve as templates for the design of vaccines against HPAI virus H5N1.

Plans:

1. We continue to elucidate mechanism of antibody-mediated viral neutralization by defining the epitopes of a panel of neutralizing and cross-neutralizing anti-H5 antibodies using a yeast display system and crystal structure of mAbs in complex with antigens.

2. We will continue to perform immune-driven escape mutant studies to generate viral escape mutants under the selection of a number of a high affinity, potent neutralizing mAbs that map to different epitopes. The mutant HA genes will be sequenced and the structural changes that resulted in neutralization escape will be determined.

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