PAST PROJECT

Synthetic Genomic Tools to Improve Influenza Virus Vaccine Production (SYNFLUENZA)

The goal of this demonstration project is to develop a technical capability to stockpile vaccine-ready influenza virus seed stocks for use in vaccine production. This process will increase the speed, reliability and capacity of vaccine production. Moreover, this technology will enable a rapid response against any emergent influenza virus. By merging the powers of high-throughput influenza genomics and synthetic biology, we will make DNA reagents for the production of vaccines against recommended strains of seasonal and pandemic strains of influenza virus. The proposed DNA reagents are clones containing inserts that can be used as a starting point for creating vaccine stocks. They will be made available to the influenza virus research and vaccine development community through the NIAID Biodefense and Emerging Infections Research Resources Repository (BEI).

This project will use JCVI synthetic genomics technology to synthesize 1000 hemagglutinin (HA) and 1000 neuraminidase (NA) genes that represent the range of protein sequence diversity observed in human, porcine, and avian infections during the period from 2005-2010. These sequence verified gene clones will be in pUC19 plasmid vectors. Additionally, this effort will demonstrate the automation of the DNA synthesis approaches previously used only to make individual DNA segments. Informatics and robotic liquid handling methods have been developed for the design and assembly of the 1.4 kb NA and 1.8 kb HA genes from synthetic oligonucleotides. We designed our HA and NA synthesis strategy based on the 2 step approach we took to synthesize a 16 Kbp mouse mitochondrial genome from synthetic oligonucleotides. In the first step we mix 8 overlapping 60 base oligos along with the enzymes needed to convert the oligos into a population of synthetic ~350 bp DNA fragments or cassettes. These fragments are cloned in Escherichia. coli, and sequence verified. In the second step, the overlapping ~350 bp DNA fragments are assembled using JCVI's chew back anneal protocol, which is also known as Gibson Assembly. Each HA molecule is comprised of 7 cassettes, and each NA is comprised of 5 cassettes. We will synthesize 8,950 ~350 bp DNA cassettes from 27,929 different ~ 60 base oligonucleotides.

Funding

This project has been funded in whole or part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under contract numbers N01-AI30071 and/or HHSN272200900007C.

Presentations

Related Research