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. 2023 Aug 15;11(8):1364.
doi: 10.3390/vaccines11081364.

Development of an Enhanced High-Yield Influenza Vaccine Backbone in Embryonated Chicken Eggs

Lizheng Guan  1 , Jihui Ping  1 , Tiago J S Lopes  1 , Shufang Fan  1 , Robert Presler  1 , Gabriele Neumann  1 , Yoshihiro Kawaoka  1   2   3
Affiliations

Development of an Enhanced High-Yield Influenza Vaccine Backbone in Embryonated Chicken Eggs

Lizheng Guan et al. Vaccines (Basel). .

Abstract

Vaccination is an efficient approach to preventing influenza virus infections. Recently, we developed influenza A and B virus vaccine backbones that increased the yield of several vaccine viruses in Madin-Darby canine kidney (MDCK) and African green monkey kidney (Vero) cells. These vaccine backbones also increased viral replication in embryonated chicken eggs, which are the most frequently used platform for influenza vaccine manufacturing. In this study, to further increase the viral titers in embryonated chicken eggs, we introduced random mutations into the 'internal genes' (i.e., all influenza viral genes except those encoding the hemagglutinin and neuraminidase proteins) of the influenza A virus high-yield virus backbone we developed previously. The randomly mutated viruses were sequentially passaged in embryonated chicken eggs to select variants with increased replicative ability. We identified a candidate that conferred higher influenza virus growth than the high-yield parental virus backbone. Although the observed increases in virus growth may be considered small, they are highly relevant for vaccine manufacturers.

Keywords: eggs; high yield; influenza A virus; vaccine.

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Conflict of interest statement

Y.K. has received speaker’s honoraria from Toyama Chemical and Astellas Inc. and grant support from Chugai Pharmaceuticals, Daiichi Sankyo Pharmaceutical, Toyama Chemical, Tauns Laboratories, Inc., Otsuka Pharmaceutical Co., Ltd., Denka Seiken Co., Ltd., and Shionogi & Co., Ltd. Y.K. and G.N. are co-founders of FluGen. The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Flow chart summarizing the selection and testing of the enhanced high-yield vaccine backbone. See the text for details.
Figure 2
Figure 2
HA and virus titers in embryonated chicken eggs of two high-yield vaccine candidates. Viruses expressing the Singapore HA and NA proteins with the genetic backbone of the parental PR8-HY or the enhanced S12- and S13-HY candidates were inoculated into embryonated chicken eggs (2 × 103 PFU/egg), and the HA (a) and virus (b) titers were measured 48 h later. Shown are the averages of three independently generated virus stocks. The titers were compared using a one-way ANOVA, followed by Tukey’s post-hoc test. * p < 0.05.
Figure 3
Figure 3
Growth curves in embryonated chicken eggs of vaccine viruses and recombinant viruses with different genetic backbones. The HA and NA genes of egg-grown Singapore (a,b), Switzerland (c,d), Kansas (e,f), and Michigan (g,h) vaccine viruses were combined with the genetic backbone of parental PR8-HY or enhanced S12-HY. For comparison, we also tested authentic egg-grown Singapore (IVR-186) (a,b), Switzerland (NIB-112) (c,d), Kansas (NYMC X-327) (e,f), and Michigan (NYMC-X275) (g,h) vaccine viruses from NIBSC. Ten-day-old embryonated chicken eggs were infected with 2 × 102 PFU of the virus. The allantoic fluids of three eggs were harvested at the indicated time points. The HA and virus titers were measured by conducting HA and plaque assays in MDCK cells, respectively.
Figure 4
Figure 4
Total viral protein and HA content of vaccine viruses and recombinant viruses with different genetic backbones. Viral protein was deglycosylated using PNGase F (New England Biolabs) and then subjected to SDS-PAGE (a,b). HA contents (d) were calculated according to the amounts of total viral protein (c) and the relative amounts of HA). The HA contents are expressed in mg per 100 eggs. Asterisks denote a significant difference between groups. The values presented are the average of three independent values ± s.d. Statistical significance was determined by multiple comparisons using a one-way ANOVA test. ** p < 0.01; *** p < 0.001; **** p < 0.0001.
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