The Source of the PB1 Gene in Influenza Vaccine Reassortants Selectively Alters the Hemagglutinin Content of the Resulting Seed Virus

Cobbin, Joanna C. A.; Verity, Erin E.; Gilbertson, Brad; Rockman, Steven; Brown, Lorena E.

doi:10.1128/jvi.02856-12

Cited by 32 publications

(54 citation statements)

References 37 publications

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“…Our own retrospective analysis of past vaccine seed strains available at bioCSL, Ltd. (8), is in agreement with other similar analyses that indicate that most of the non-HA and NA genes are derived from the high-growth parent. However, the seasonal PB1 gene dominates in progeny viruses in about 50 to 60% of reassortment events.…”

supporting

confidence: 73%

Influenza Virus PB1 and Neuraminidase Gene Segments Can Cosegregate during Vaccine Reassortment Driven by Interactions in the PB1 Coding Region

Cobbin

Ong

Verity

et al. 2014

J Virol

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Egg-grown influenza vaccine yields are maximized by infection with a seed virus produced by "classical reassortment" of a seasonal isolate with a highly egg-adapted strain. Seed viruses are selected based on a high-growth phenotype and the presence of the seasonal hemagglutinin (HA) and neuraminidase (NA) surface antigens. Retrospective analysis of H3N2 vaccine seed viruses indicated that, unlike other internal proteins that were predominantly derived from the high-growth parent A/Puerto Rico/8/34 (PR8), the polymerase subunit PB1 could be derived from either parent depending on the seasonal strain. We have recently shown that A/Udorn/307/72 (Udorn) models a seasonal isolate that yields reassortants bearing the seasonal PB1 gene. This is despite the fact that the reverse genetics-derived virus that includes Udorn PB1 with Udorn HA and NA on a PR8 background has inferior growth compared to the corresponding virus with PR8 PB1. Here we use competitive plasmid transfections to investigate the mechanisms driving selection of a less fit virus and show that the Udorn PB1 gene segment cosegregates with the Udorn NA gene segment. Analysis of chimeric PB1 genes revealed that the coselection of NA and PB1 segments was not directed through the previously identified packaging sequences but through interactions involving the internal coding region of the PB1 gene. This study identifies associations between viral genes that can direct selection in classical reassortment for vaccine production and which may also be of relevance to the gene constellations observed in past antigenic shift events where creation of a pandemic virus has involved reassortment. IMPORTANCEInfluenza vaccine must be produced and administered in a timely manner in order to provide protection during the winter season, and poor-growing vaccine seed viruses can compromise this process. To maximize vaccine yields, manufacturers create hybrid influenza viruses with gene segments encoding the surface antigens from a seasonal virus isolate, important for immunity, and others from a virus with high growth properties. This involves coinfection of cells with both parent viruses and selection of dominant progeny bearing the seasonal antigens. We show that this method of creating hybrid viruses does not necessarily select for the best yielding virus because preferential pairing of gene segments when progeny viruses are produced determines the genetic makeup of the hybrids. This not only has implications for how hybrid viruses are selected for vaccine production but also sheds light on what drives and limits hybrid gene combinations that arise in nature, leading to pandemics.

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confidence: 73%

Influenza Virus PB1 and Neuraminidase Gene Segments Can Cosegregate during Vaccine Reassortment Driven by Interactions in the PB1 Coding Region

Cobbin

Ong

Verity

et al. 2014

J Virol

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“…ratios (Tables 1 and 2). In another study, it was shown that the PB1 segment derived from the seasonal H3N2 virus A/Udorn/307/72 (Udorn) helped to improve the PR8-based 5+3 IVS carrying HA/NA/ PB1 Undorn by a fourfold increase of the HA content per virion, even though this IVS was replicating less efficiently than the corresponding 6+2 IVS (Cobbin et al, 2013). Yet our results, which are consistent with those of a previous study by Harvey et al (2011), demonstrate that the HA yield of our IVSs rather depends on virus growth rate than on HA content per viral particle.…”

Section: Discussionmentioning

confidence: 99%

“…Given that virus neutralization requires antibodies that recognize specific epitopes on the HA protein, a high HA content per virion allows to increase the overall amount of produced vaccines (Cobbin et al, 2013). We therefore sought to rule out the possibility that enhanced replication of 5+2+1 IVSs resulted in virus particles with reduced HA content.…”

Section: Pb1 Gi Enhances the Replication Of Pr8-derived Ivss With DIVmentioning

confidence: 99%

“…4)]. The combined data led us to conclude that the additional insertion of the PB1 Gi segment provided a significant growth advantage in recombinant PR8-derived IVSs containing heterologous HA/NA segments, while it was neutral in a few other cases.Similar HA content per viral particle in PR8-based 5 1 2 1 1 and 6 1 2 IVSsGiven that virus neutralization requires antibodies that recognize specific epitopes on the HA protein, a high HA content per virion allows to increase the overall amount of produced vaccines (Cobbin et al, 2013). We therefore sought to rule out the possibility that enhanced replication of 5+2+1 IVSs resulted in virus particles with reduced HA content.…”

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The PB1 segment of an influenza A virus H1N1 2009pdm isolate enhances the replication efficiency of specific influenza vaccine strains in cell culture and embryonated eggs

Mostafa

Kanrai

Ziebuhr

et al. 2016

Journal of General Virology

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Influenza vaccine strains (IVSs) contain the haemagglutinin (HA) and neuraminidase (NA) genome segments of relevant circulating strains in the genetic background of influenza A/ PR/8/1934 virus (PR8). Previous work has shown that the nature of the PB1 segment may be a limiting factor for the efficient production of IVSs. Here, we showed that the PB1 segment (PB1 Gi ) from the 2009 pandemic influenza A virus (IAV) A/Giessen/06/2009 (Gi wt, H1N1pdm) may help to resolve (some of) these limitations. We produced a set of recombinant PR8-derived viruses that contained (i) the HA and NA segments from representative IAV strains (H3N2, H5N1, H7N9, H9N2); (ii) the PB1 segment from PR8 or Gi wt, respectively; and (iii) the remaining five genome segments from PR8. Viruses containing the PB1 Gi segment, together with the heterologous HA/NA segments and five PR8 segments (5+2+1), replicated to higher titres compared with their 6+2 counterparts containing six PR8 segments and the equivalent heterologous HA/NA segments. Compared with PB1 PR8 -containing IVSs, viruses with the PB1 Gi segment replicated to higher or similar titres in both cell culture and embryonated eggs, most profoundly IVSs of the H5N1 and H7N9 subtype, which are known to grow poorly in these systems. IVSs containing either the PB1 Gi or the cognate PB1 segment of the respective specific HA/NA donor strain showed enhanced or similar virus replication levels. This study suggests that substitution of PB1 PR8 with the PB1 Gi segment may greatly improve the large-scale production of PR8-derived IVSs, especially of those known to replicate poorly in vitro. INTRODUCTIONInfluenza viruses belong to the family Orthomyxoviridae and are classified into three genera termed Influenza virus A, B and C. Among these, influenza A viruses (IAVs) pose the greatest threat to human and animal health. They contain eight segments of negative-sense ssRNA (Ritchey et al., 1976;Shaw & Palese, 2013). The eight genomic segments encode at least 10 viral proteins, comprising the three subunits of the viral RNA polymerase complex [polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1) and polymerase acidic protein (PA)], the surface glycoproteins [haemagglutinin (HA) and neuraminidase (NA)], the nucleoprotein (NP), the matrix protein (M1), the ion channel protein (M2), the non-structural protein 1 (NS1) and the nuclear export protein (NEP). IAVs are genetically diverse, with new strains rapidly emerging by point mutations introduced by the low-fidelity viral RNA replicase (potentially resulting in antigenic drift if changes occur in the HA and/or NA coding sequences and affect important antigenic epitopes) or by genetic reassortment of genomic segments (potentially leading to antigenic shift if antigenically distant HA and NA segments are incorporated). Minor and major antigenic changes in the HA and NA proteins of circulating IAV are important driving forces for the emergence of new strains causing epidemics or even major pandemics (Neumann et al., 2009;Peng et al., 2014;Smith...

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“…PB1 from a seasonal strain or with targeted mutations has been shown to improve growth or HA yield of H3 reassortants (Cobbin et al, 2013;Plant et al, 2012); however, mixed results have been observed for H5 reassortants (Abt et al, 2011;Rudneva et al, 2007;Wanitchang et al, 2010). All three polymerase segments reassort together more frequently in experimental settings than natural reassortment, suggesting gene constellation effects (Macken et al, 2006;Varich et al, 2008;Wille et al, 2013).…”

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confidence: 99%

Chimeric neuraminidase and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus

Plant

2015

Journal of General Virology

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We previously showed that a mutated PB1 gene improved the growth kinetics of a H3N2 influenza reassortant. Here, we showed that the same mutations improved the growth kinetics of a virus containing the A/Vietnam/1203/2004 (H5N1) haemagglutinin and neuraminidase (NA). Total protein yield and NA activity were increased when a chimeric NA was included. These increases indicated that the synergistic effect was due to the gene constellation containing both the altered PB1 gene and the chimeric NA gene.The object of this work was to improve the genomic backbone segments used in vaccine candidate viruses whilst avoiding detrimental gene constellation effects. It is not known what attributes from each segment affect the gene constellation or reassortment, but it is desirable that vaccine seed strains containing contemporary haemagglutinin (HA) and neuraminidase (NA) segments in the context of known backbone segments can be recovered. It is also desirable that those strains replicate well and that satisfactory protein yields can be harvested. We have previously shown that five mutations to the PB1 gene of the high-growth backbone virus PR/8 improved the growth kinetics of a reassortant containing the HA and NA segments from an H3N2 virus (Plant et al., 2012). These mutations (G180E, S216G, S361R, Q621R and N654S) were not selected based on any structural or functional information. The five mutations made to the PB1 gene (PB1 m5 ) were based on sequence variations found in vaccine strain reassortants. We have also described the improved growth and protein yield of a reassortant (Table 1) and all the viruses produced large plaques on MDCK cells (Fig. 1a). The longer stalk region of the H5N1 NA has been shown to contribute to pathogenicity (Zhou et al., 2009). Here, we confirmed that removing this region reduced pathogenicity, with or without the PB1 M5 allele, by determining mean death time (MDT). Virus particles (10 6 ) were inoculated into the allantoic fluid of eight eggs, which were then incubated at 35 u C and candled at 12 h intervals. Eggs were scored as alive or dead and the time of death recorded. MDT was obtained by multiplying the number of embryos dead at each interval post-inoculation by the hours post-inoculation and dividing by the total number of embryos inoculated. The MDT range was similar for all viruses (Table 1).Optimal polymerase activity is required for efficient replication (Giesendorf et al., 1986;Li et al., 2009;Ngai et al., 2013). PB1 from a seasonal strain or with targeted mutations has been shown to improve growth or HA yield of H3 reassortants (Cobbin et al., 2013;Plant et al., 2012); however, mixed results have been observed for H5 reassortants (Abt et al., 2011;Rudneva et al., 2007;Wanitchang et al., 2010). All three polymerase segments reassort together more frequently in experimental settings than natural reassortment, suggesting gene constellation effects (Macken et al., 2006;Varich et al., 2008;Wille et al., 2013). Here, we examined the growth of H5 reassortants with the PB1 m5 allele w...

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The Source of the PB1 Gene in Influenza Vaccine Reassortants Selectively Alters the Hemagglutinin Content of the Resulting Seed Virus

Cited by 32 publications

References 37 publications

Influenza Virus PB1 and Neuraminidase Gene Segments Can Cosegregate during Vaccine Reassortment Driven by Interactions in the PB1 Coding Region

Influenza Virus PB1 and Neuraminidase Gene Segments Can Cosegregate during Vaccine Reassortment Driven by Interactions in the PB1 Coding Region

The PB1 segment of an influenza A virus H1N1 2009pdm isolate enhances the replication efficiency of specific influenza vaccine strains in cell culture and embryonated eggs

Chimeric neuraminidase and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus

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