Abstract
Candidate vaccine viruses (CVVs) for seasonal influenza A virus are made by reassortment of the antigenic virus with an egg-adapted strain, typically A/Puerto Rico/8/34 (PR8). Many 2009 A(H1N1) pandemic (pdm09) high-growth reassortants (HGRs) selected this way contain pdm09 segment 2 in addition to the antigenic genes. To investigate this, we made CVV mimics by reverse genetics (RG) that were either 6 : 2 or 5 : 3 reassortants between PR8 and two pdm09 strains, A/California/7/2009 (Cal7) and A/England/195/2009, differing in the source of segment 2. The 5 : 3 viruses replicated better in MDCK-SIAT1 cells than the 6 : 2 viruses, but the 6 : 2 CVVs gave higher haemagglutinin (HA) antigen yields from eggs. This unexpected phenomenon reflected temperature sensitivity conferred by pdm09 segment 2, as the egg HA yields of the 5 : 3 viruses improved substantially when viruses were grown at 35 °C compared with 37.5 °C, whereas the 6 : 2 virus yields did not. However, the authentic 5 : 3 pdm09 HGRs, X-179A and X-181, were not markedly temperature sensitive despite their PB1 sequences being identical to that of Cal7, suggesting compensatory mutations elsewhere in the genome. Sequence comparisons of the PR8-derived backbone genes identified polymorphisms in PB2, NP, NS1 and NS2. Of these, PB2 N701D affected the temperature dependence of viral transcription and, furthermore, improved and drastically reduced the temperature sensitivity of the HA yield from the 5 : 3 CVV mimic. We conclude that the HA yield of pdm09 CVVs can be affected by an epistatic interaction between PR8 PB2 and pdm09 PB1, but that this can be minimized by ensuring that the backbones used for vaccine manufacture in eggs contain PB2 701D.
Highlights
Worldwide, annual influenza epidemics result in three to five million cases of severe illness, and 250,000 to 500,000 deaths [1]
Introduction shown that candidate vaccine viruses (CVVs) 5:3 reassortants containing a pdm09 segment 2 and glycoproteins of avian H5N1 and H7N9 viruses give higher yields than their respective 5:3 viruses containing the indigenous WT segment 2, suggesting a particular growth advantage conferred to CVVs by the pdm09 segment 2 [22]
The expectation, based on empirical evidence from existing high-growth reassortants (HGRs) as well as from published work that used reverse genetics (RG) methods [14,15,16,17,18,19,20,21,22], was that the 5:3 reassortants would grow better than the 6:2 viruses
Summary
Annual influenza epidemics result in three to five million cases of severe illness, and 250,000 to 500,000 deaths [1]. Seasonal vaccine production techniques rely on classical reassortment to generate viruses with good growth properties in embryonated hens’ eggs, the major manufacturing substrate This involves co-infecting eggs with the antigenic (vaccine strain) virus of choice along with a high yielding (“donor”) virus already adapted to growth in eggs. Segment packaging signals of the glycoprotein genes are known to influence yield [14, 25,26,27,28,29,30,31,32] and it has been demonstrated for H3N2 subtype 5:3 reassortants that the NA and PB1 segments co-segregate, driven by interactions in the coding region of segment 2 [17, 22]. A better understanding of the molecular basis for the effects of vaccine strain-derived segment 2s on growth of reassortant
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