Development of high-yield influenza A virus vaccine viruses

Abstract

Vaccination is one of the most cost-effective ways to prevent infection. Influenza vaccines propagated in cultured cells are approved for use in humans, but their yields are often suboptimal. Here, we screened A/Puerto Rico/8/34 (PR8) virus mutant libraries to develop vaccine backbones (defined here as the six viral RNA segments not encoding haemagglutinin and neuraminidase) that support high yield in cell culture. We also tested mutations in the coding and regulatory regions of the virus, and chimeric haemagglutinin and neuraminidase genes. A combination of high-yield mutations from these screens led to a PR8 backbone that improved the titres of H1N1, H3N2, H5N1 and H7N9 vaccine viruses in African green monkey kidney and Madin-Darby canine kidney cells. This PR8 backbone also improves titres in embryonated chicken eggs, a common propagation system for influenza viruses. This PR8 vaccine backbone thus represents an advance in seasonal and pandemic influenza vaccine development.

Figure 1. Flow chart summarizing the selection and testing of PR8-HY.

Details are described in the text.

Figure 2. Growth kinetics and HA titres of HY#1–7 high-yield candidates in Vero cells.

(a) Growth kinetics and HA titres of high-yield candidates in Vero cells. Vero cells were infected in triplicate with the indicated viruses at a multiplicity of infection (MOI) of 0.005 and incubated at 37 °C. Supernatants were collected at the indicated time points, and the virus titres were determined by plaque assays in MDCK cells. In parallel, we determined the HA titres of the collected supernatants by performing HA assays. (b) Effect of the C4U promoter mutation in the viral polymerase genes on viral growth kinetics and HA titres. Shown is the comparison of viruses possessing the parental UW-PR8 backbone (UW-PR8_Indo09), the HY#1 backbone (HY#1_Indo09), or the HY#1 backbone with C4U mutations in the PB2, PB1 and PA genes (HY#1+C4U_Indo09). Experiments were carried out as described in a. The values presented are the average of three independent experiments±s.d.

Figure 3. Growth kinetics and HA titres of HA/NA chimeric viruses in Vero cells.

(a) Growth kinetics and HA titres of UW-PR8-based viruses with wild-type or chimeric Indo09 HA and NA genes. (b) Growth kinetics and HA titres of viruses possessing the UW-PR8 backbone in combination with Indo09 HA and NA genes, or the PR8-HY backbone in combination with wild-type or chimeric Indo09 HA and NA genes. Experiments were carried out as described in the legend to Fig. 2. The values presented are the average of three independent experiments±s.d.

Figure 4. Evaluation of PR8-HY backbone vaccine candidates propagated in Vero cells.

Growth kinetics and HA titres of UW-PR8- and PR8-HY-based viruses encoding the wild type, or wild-type and chimeric HA and NA segments of the A/Vietnam/1203/2004 (VN04, H5N1) (a), A/Hubei/1/2010 (Hubei10, H5N1) (b), A/Egypt/N03072/2010 (Egypt10, H5N1) (c), A/Indonesia/5/2005 (Indo05, H5N1) (d) or A/Anhui/1/2013 (Anhui13, H7N9) (e) viruses. Panels (f) and (g) show a comparison of current seasonal H1N1 and H3N2 vaccine viruses (X-181 and X-223A, respectively) with PR8-HY backbone viruses possessing wild-type or chimeric HA and NA segments derived from X-181 or X-223A viruses. Experiments were carried out as described in the legend to Fig. 2, with the exception of those involving X-181 and X-223A viruses, which were inoculated at an MOI of 0.1. The values presented are the average of three independent experiments±s.d.

Figure 5. Evaluation of PR8-HY vaccine candidate viruses propagated in MDCK cells.

Growth kinetics and HA titres of UW-PR8- and PR8-HY-based viruses encoding the wild-type, or wild-type and chimeric HA and NA segments of the A/chicken/Indonesia/NC/2009 (Indo09, H5N1) (a), A/Vietnam/1203/2004 (VN04, H5N1) (b), A/Hubei/1/2010 (Hubei10, H5N1) (c), A/Egypt/N03072/2010 (Egypt10, H5N1) (d), A/Indonesia/5/2005 (Indo05, H5N1) (e), or A/Anhui/1/2013 (Anhui13, H7N9) (f) viruses. Panels (g) and (h) show a comparison of current seasonal H1N1 and H3N2 vaccine viruses (X-181 and X-223A, respectively) with PR8-HY backbone viruses possessing wild-type or chimeric HA and NA segments derived from X-181 or X-223A viruses. MDCK cells were infected in triplicate with the indicated viruses at an MOI of 0.001 and incubated at 37 °C; otherwise, experiments were carried out as described in the legend to Fig. 2. The values presented are the average of three independent experiments±s.d.

Figure 6. Evaluation of PR8-HY vaccine candidate viruses propagated in embryonated chicken eggs.

Growth kinetics and HA titres of UW-PR8- and PR8-HY-based viruses encoding the wild-type, or wild-type and chimeric HA and NA segments of the A/chicken/Indonesia/NC/2009 (Indo09, H5N1) (a), A/Vietnam/1203/2004 (VN04, H5N1) (b), A/Hubei/1/2010 (Hubei10, H5N1) (c), A/Egypt/N03072/2010 (Egypt10, H5N1) (d), A/Indonesia/5/2005 (Indo05, H5N1) (e), or A/Anhui/1/2013 (Anhui13, H7N9) (f) viruses. Panels (g) and (h) show a comparison of current seasonal H1N1 and H3N2 vaccine viruses (X-181 and X-223A, respectively) with PR8-HY backbone viruses possessing wild-type or chimeric HA and NA segments derived from X-181 or X-223A viruses. Ten-day-old embryonated chicken eggs (four per virus) were inoculated with 2 × 10³ PFU of the respective viruses and incubated at 35 °C for the indicated periods of time. The values presented are the average of three independent experiments±s.d.

Figure 7. Evaluation of the total viral protein and HA content of PR8-HY candidate vaccine viruses.

Total viral protein yield of Vero cell- (a) or egg-grown (d), sucrose gradient-purified virus samples. The total protein content of virus concentrates was measured by using the Pierce BCA assay kit (Thermo Fisher) according to the manufacturer's instructions. SDS–PAGE analyses of virus samples from Vero cells (b) or embryonated chicken eggs (e). Virus concentrates were deglycosylated with PNGase (PNFase F+) or left untreated (PNGase F−). HA contents of Vero-; (c) and egg-grown (f) viruses. The HA contents were calculated based on the total viral protein amounts (Fig. 7a,d) and the relative amounts of HA (Fig. 7b,e); for details, see Study Design. The HA contents are expressed in mg l⁻¹ (for Vero cell-grown viruses) or mg per 100 eggs (for viruses grown in embryonated chicken eggs). Asterisks indicate a significant difference. The values presented are the average of three independent experiments±s.d. P-values were calculated by using Tukey's post-hoc test, comparing the total viral protein yield and HA content of wild-type viruses with that of recombinant high-yield vaccine viruses; ***P<0.005.