The source of the PB1 gene in influenza vaccine reassortants selectively alters the hemagglutinin content of the resulting seed virus

Abstract

The yields of egg-grown influenza vaccines are maximized by the production of a seed strain using a reassortment of the seasonal influenza virus isolate with a highly egg-adapted strain. The seed virus is selected based on high yields of viral hemagglutinin (HA) and expression of the surface antigens from the seasonal isolate. The remaining proteins are usually derived from the high-growth parent. However, a retrospective analysis of vaccine seeds revealed that the seasonal PB1 gene was selected in more than 50% of reassortment events. Using the model seasonal H3N2 virus A/Udorn/307/72 (Udorn) virus and the high-growth A/Puerto Rico/8/34 (PR8) virus, we assessed the influence of the source of the PB1 gene on virus growth and vaccine yield. Classical reassortment of these two strains led to the selection of viruses that predominantly had the Udorn PB1 gene. The presence of Udorn PB1 in the seed virus, however, did not result in higher yields of virus or HA compared to the yields in the corresponding seed virus with PR8 PB1. The 8-fold-fewer virions produced with the seed virus containing the Udorn PB1 were somewhat compensated for by a 4-fold increase in HA per virion. A higher HA/nucleoprotein (NP) ratio was found in past vaccine preparations when the seasonal PB1 was present, also indicative of a higher HA density in these vaccine viruses. As the HA viral RNA (vRNA) and mRNA levels in infected cells were similar, we propose that PB1 selectively alters the translation of viral mRNA. This study helps to explain the variability of vaccine seeds with respect to HA yield.

Fig 1

The genetic makeup of reassortant viruses affects HA titer and viral growth. The hemagglutination titers of individual eggs infected with a constant dose (100 PFU) of the wild-type (wt) virus parents PR8 and Udorn were compared to those of the PR8(Ud-HA,NA,PB1) virus selected by classical reassortment of the two parental viruses (A). The hemagglutination titers (B) and infectious virus titers, determined by plaque formation in MDCK cells (C), were measured in eggs infected with a constant dose (100 PFU) of the reverse genetics-derived viruses corresponding to the PR8 and Udorn parent viruses and additional viruses containing Udorn HA and NA with or without the Udorn PB1, PB2, or PA (with the remaining genes originating from PR8). The titers of viral particles (D), measured using copy numbers of M vRNA in the allantoic fluid samples used in the assay whose results are shown in panel C, were determined by quantitative RT-PCR as a surrogate. The yields of infectious virus from individual MDCK cell cultures infected with 100 PFU of virus were determined by plaque assay of cell culture supernatants (E). In panels A, B, C, and E, each symbol represents the titer of an individual egg or culture supernatant and the line is the geometric mean titer. In panel D, each bar represents the mean particle number of all the eggs in that group and the error bar shows the standard error of the mean.

Fig 2

The composition of the RNP complex affects the intrinsic polymerase activity in a minigenome assay but not the production of vRNA or viral mRNA in infected cells. (A) Minigenome assays were performed in 293T cells transfected with the pCAGGS-BLA reporter gene and pHW2000 plasmids that each expressed a component of the influenza virus RNP complex. The polymerase activities of the PR8 and Udorn RNP complexes were measured, in addition to those of the complexes expressing the Udorn NP or PB1 with the remaining RNP proteins from PR8, referred to as PR8(Ud-NP) RNP and PR8(Ud-PB1) RNP, respectively. Each bar represents the mean of three independent experiments, and the error bars show the standard errors of the means, each normalized to the activity of the PR8 RNP complex within each experiment. (B to D) MDCK cells were infected with PR8(Ud-HA,NA) virus (white) or PR8(Ud-HA,NA,PB1) virus (gray) at an MOI of 3 PFU per cell. Total RNA was extracted from 1 × 10⁶ infected cells at 0, 4, 8, and 24 h after a 1-h virus absorption period, and the copy numbers of the M vRNA (B) and the viral mRNA resulting from the transcription of the M gene (M1 and M2 mRNA) (C) and HA mRNA (D) were assessed by quantitative RT-PCR. Each bar represents the geometric mean of three independent experiments, and the error bars represent the standard errors of the means.

Fig 3

Viral protein expression in cells infected with viruses differing in the origin of the PB1 protein. Aliquots of 1 × 10⁵ MDCK cells infected with PR8(Ud-HA,NA) virus or PR8(Ud-HA,NA,PB1) virus from the preparations used in the experiments whose results are shown in Figure 2B, C, and D were disrupted and separated on 4 to 20% Tris-glycine gels under nonreducing conditions. Proteins were transferred to PVDF membranes and probed with anti-Udorn HA and anti-M1 monoclonal antibodies. pi, postinfection. (A). The relative contents of HA (B) and M1 (C) proteins were determined by densitometry of stained bands and analyzed using the ImageQuant TL software. White bars, PR8(Ud-HA,NA) virus; gray bars, PR8(Ud-HA,NA,PB1) virus. Each bar represents the geometric mean of two independent experiments, and the error bars represent the standard errors of the means.

Fig 4

Western blots used for the determination of relative viral protein contents of samples reported in Tables 3 and 4. (A) Eggs were inoculated with 100 PFU of either PR8(Ud-HA,NA) or PR8(Ud-HA,NA,PB1). Allantoic fluid was clarified by ultracentrifugation, and the virus was pelleted at 31,000 × g and then resuspended in PBS. Tris-glycine gels (4 to 20%) were loaded with 20,000 HAU of virus sample and separated by SDS-PAGE under reducing conditions. Proteins were transferred to a PVDF membrane and probed with either anti-NP or anti-HA2 monoclonal antibodies. The relative protein contents were determined by densitometry of stained bands analyzed using the ImageQuant TL software and are presented in Table 3. (B) Monovalent split virus vaccine preparations were deglycosylated, and samples containing 21 μg of viral protein, as determined by Lowry assay, were separated on 10% bis-Tris gels under reducing conditions in the presence of NuPAGE antioxidant. Proteins were transferred to a PVDF membrane and probed with either anti-NP or anti-HA2 monoclonal antibodies. The relative protein contents were determined by densitometry of stained bands analyzed using the ImageQuant TL software and are presented in Table 4.