Rapid generation of pandemic influenza virus vaccine candidate strains using synthetic DNA

Abstract

Background: Vaccination is considered the most effective means of reducing influenza burden. The emergence of H5N1 and pandemic spread of novel H1N1/2009 viruses reinforces the need to have strategies in place to rapidly develop seed viruses for vaccine manufacture.

Methods: Candidate pandemic vaccine strains consisting of the circulating strain haemagglutinin (HA) and neuraminidase (NA) in an A/PR/8/34 backbone were generated using alternative synthetic DNA approaches, including site-directed mutagenesis of DNA encoding related virus strains, and rapid generation of virus using synthetic DNA cloned into plasmid vectors.

Results: Firstly, synthetic A/Bar Headed Goose/Qinghai/1A/2005 (H5N1) virus was generated from an A/Vietnam/1194/2004 template using site-directed mutagenesis. Secondly, A/Whooper Swan/Mongolia/244/2005 (H5N1) and A/California/04/09 (H1N1) viruses were generated using synthetic DNA encoding the viral HA and NA genes. Replication and antigenicity of the synthetic viruses were comparable to that of the corresponding non-synthetic viruses.

Conclusions: In the event of an influenza pandemic, the use of these approaches may significantly reduce the time required to generate and distribute the vaccine seed virus and vaccine manufacture. These approaches also offer the advantage of not needing to handle wild-type virus, potentially diminishing biocontainment requirements.

Figure 1

Steps required for the generation of influenza virus vaccine candidates using reverse genetics. In the current method (A), viral HA and neuraminidase (NA) are isolated from wild‐type virus, cloned into plasmid DNA for reverse genetics, and the polybasic region is removed before the candidate strain is produced using reverse genetics and distributed to vaccine manufacturers. Alternatively, vaccine manufacturers may produce plasmids for reverse genetics on‐site by site‐directed mutagenesis of the template DNA of a related strain (B), or using synthetic DNA encoding the HA and NA of the vaccine candidate strain with the polybasic region already removed (C).

Figure 2

Growth comparison of reassortant and reverse genetics swine influenza viruses in eggs. Bars represent HA activity in allantoic fluid pooled from 36 eggs for each virus. Results are from a single experiment representative of two separate growth comparisons and data accumulated from serial passage of each virus up to 4 times in eggs.