Identification of Influenza A/PR/8/34 Donor Viruses Imparting High Hemagglutinin Yields to Candidate Vaccine Viruses in Eggs

Abstract

One of the important lessons learned from the 2009 H1N1 pandemic is that a high yield influenza vaccine virus is essential for efficient and timely production of pandemic vaccines in eggs. The current seasonal and pre-pandemic vaccine viruses are generated either by classical reassortment or reverse genetics. Both approaches utilize a high growth virus, generally A/Puerto Rico/8/1934 (PR8), as the donor of all or most of the internal genes, and the wild type virus recommended for inclusion in the vaccine to contribute the hemagglutinin (HA) and neuraminidase (NA) genes encoding the surface glycoproteins. As a result of extensive adaptation through sequential egg passaging, PR8 viruses with different gene sequences and high growth properties have been selected at different laboratories in past decades. The effect of these related but distinct internal PR8 genes on the growth of vaccine viruses in eggs has not been examined previously. Here, we use reverse genetics to analyze systematically the growth and HA antigen yield of reassortant viruses with 3 different PR8 backbones. A panel of 9 different HA/NA gene pairs in combination with each of the 3 different lineages of PR8 internal genes (27 reassortant viruses) was generated to evaluate their performance. Virus and HA yield assays showed that the PR8 internal genes influence HA yields in most subtypes. Although no single PR8 internal gene set outperformed the others in all candidate vaccine viruses, a combination of specific PR8 backbone with individual HA/NA pairs demonstrated improved HA yield and consequently the speed of vaccine production. These findings may be important both for production of seasonal vaccines and for a rapid global vaccine response during a pandemic.

Biosafety risk assessment for production of candidate vaccine viruses to protect humans from zoonotic highly pathogenic avian influenza viruses

“…the phenotypic character of avirulence in chickens ( Table 2). 33 Taken together, these studies strongly support the absolute necessity of a basic amino acid cluster or insertion of additional amino acids at the cleavage site to impart high virulence for chickens to reassortant viruses with PR8 genetic background.…”

“…The numerous CVVs developed to protect against H5 and H7 HPAI virus subtypes, comprising diverse HA and NA genetic lineages (Eurasian and North American) and divergent genetic clades (Table ), were produced by reverse genetic‐engineered reassortment with a human virus adapted for optimal growth in eggs, usually A/Puerto Rico/8/34 (PR8) or its derivatives . Despite extensive genetic diversity in the HA/NA and some variation among backbone virus genes, all CVVs and veterinary vaccine viruses shared the phenotypic character of avirulence in chickens (Table ) . Taken together, these studies strongly support the absolute necessity of a basic amino acid cluster or insertion of additional amino acids at the cleavage site to impart high virulence for chickens to reassortant viruses with PR8 genetic background.…”

Pathogenicity testing of influenza candidate vaccine viruses in the ferret model

“…Parallel work that can occur further downstream of the safety testing of the CVV involves analysis of HA yield and further improvements in growth characteristics necessary for large-scale expansion and stockpiling. Specifically, alternate PR/8 internal gene segments with different characteristics can be combined with the HA and NA from the CVV in order to optimize yield through different gene segment interactions (Johnson et al, 2015). In addition, further passaging in eggs is another approach to elicit egg-adapted changes that can improve yield while maintaining antigenicity similarity to parental virus (Ridenour et al, 2015a).…”

Reassortant viruses for influenza vaccines: is it time to reconsider their genome structures?

“…Thus, the majority of NP-specific T-cell epitopes of currently circulating viruses could be incorporated without a loss of seed virus yield. Moreover, a recent study suggested that the surface proteins, HA and NA, rather than internal and non-structural proteins, had the strongest impact on reassortant growth properties [15]. We therefore strongly recommend that the wild-type NP gene be included in the genome of reassortant viruses generated for IIV.…”