Synthetic Generation of Influenza Vaccine Viruses for Rapid Response to Pandemics

Abstract

During the 2009 H1N1 influenza pandemic, vaccines for the virus became available in large quantities only after human infections peaked. To accelerate vaccine availability for future pandemics, we developed a synthetic approach that very rapidly generated vaccine viruses from sequence data. Beginning with hemagglutinin (HA) and neuraminidase (NA) gene sequences, we combined an enzymatic, cell-free gene assembly technique with enzymatic error correction to allow rapid, accurate gene synthesis. We then used these synthetic HA and NA genes to transfect Madin-Darby canine kidney (MDCK) cells that were qualified for vaccine manufacture with viral RNA expression constructs encoding HA and NA and plasmid DNAs encoding viral backbone genes. Viruses for use in vaccines were rescued from these MDCK cells. We performed this rescue with improved vaccine virus backbones, increasing the yield of the essential vaccine antigen, HA. Generation of synthetic vaccine seeds, together with more efficient vaccine release assays, would accelerate responses to influenza pandemics through a system of instantaneous electronic data exchange followed by real-time, geographically dispersed vaccine production.

Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology

“…To date, there have been a handful of moonshot demonstrations such as the complete synthesis of an entire yeast chromosome (Annaluru et al 2014), an entire bacterial genome (Gibson et al 2008a), and the subsequent synthesis of a minimal bacterial genome (Hutchison et al 2016), which illustrate the use of synthetic DNA and the capabilities of existing gene synthesis methods to accomplish largescale synthetic biology efforts. These examples, when combined with numerous projects in which synthetic DNA has been used to evaluate functional biological components (Salis et al 2009;Callura et al 2012;Brophy and Voigt 2016), create synthetic vaccines (Dormitzer et al 2013), and construct synthetic genetic circuits (Salis et al 2009;Sowa et al 2015;Nielsen et al 2016;Rubens et al 2016) and when applied to wholesale genomic editing (Wang et al 2009;Doudna and Charpentier 2014), point to a future where the nuances of biological function can in part be understood by the design, synthesis, and assay of interchangeable synthetic components akin to synthetic drug development.…”

“…These methods usually immobilize MutS to a solid matrix material and then purify column-bound (error-containing) DNA sequences from unbound material (error-reduced). Error-containing heteroduplex DNA can also be sieved using enzymes that recognize and cut the DNA duplex at the site of the base mismatch (Young and Dong 2004;Fuhrmann et al 2005;Kosuri et al 2010;Saaem et al 2012;Dormitzer et al 2013). The use of endonuclease enzymes (or enzyme cocktails), which recognize and cleave DNA heteroduplexes at the sites of mismatches, has been shown to be highly effective at reducing synthesis-related errors in synthetic genes allowing for time and material savings such that in some cases the treated genes can be used directly in functional assays without cloning and sequence verification (Dormitzer et al 2013).…”

“…Error-containing heteroduplex DNA can also be sieved using enzymes that recognize and cut the DNA duplex at the site of the base mismatch (Young and Dong 2004;Fuhrmann et al 2005;Kosuri et al 2010;Saaem et al 2012;Dormitzer et al 2013). The use of endonuclease enzymes (or enzyme cocktails), which recognize and cleave DNA heteroduplexes at the sites of mismatches, has been shown to be highly effective at reducing synthesis-related errors in synthetic genes allowing for time and material savings such that in some cases the treated genes can be used directly in functional assays without cloning and sequence verification (Dormitzer et al 2013). These methods are a relatively straightforward and cost-effective approach for sieving error-containing sequences from synthetic DNA and can reduce the observed error rate from 1/100 bases to 1/10,000 bases (Dormitzer et al 2013;Kosuri and Church 2014).…”

Delivering vaccines to the people who need them most

“…During the same time frame, the synthetic HA gene from the H7N9 strain of influenza virus was used in the self-amplifying mRNA system, which is an entirely synthetic RNA vaccine that is delivered by lipid nanoparticles [25,26]. After merely one month, the vaccine was ready for immunization and analysis in animal models of infection [27].…”

“…In 2013, Venter's laboratory synthesized synthetic DNAs that encoded the HA and NA antigens of the H7N9 strain, which were used to transfect cells, together with linear plasmids encoding the other six RNA segments of the influenza virus genome [25]. During the same time frame, the synthetic HA gene from the H7N9 strain of influenza virus was used in the self-amplifying mRNA system, which is an entirely synthetic RNA vaccine that is delivered by lipid nanoparticles [25,26].…”