Effectiveness of Whole, Inactivated, Low Pathogenicity Influenza A(H7N9) Vaccine against Antigenically Distinct, Highly Pathogenic H7N9 Virus

The recent emergence of highly pathogenic influenza A(H7N9) variants poses a great risk to humans. We show that ferrets vaccinated with low pathogenicity H7N9 virus vaccine do not develop severe symptoms after infection with an antigenically distinct, highly pathogenic H7N9 virus. These results demonstrate the protective benefits of this H7N9 vaccine.


Vaccine Preparation
The HK125-HYPR8 virus was propagated in 10-day-old embryonated chicken eggs. The viruses in the allantoic fluids were inactivated with 0.1% β-propiolactone (final concentration) at 4°C overnight and then purified through ultracentrifugation by using a linear 20%-50% (w/v) sucrose gradient. The HA amount of purified virus was calculated based on the intensities of the viral protein bands separated on a 4%-12% (wt/vol) NuPAGE Bis-Tris gel (Thermo Fisher Scientific) and the amount of total viral proteins was determined by using a Pierce BCA Protein assay kit (Thermo Fisher Scientific).

Animal Experiments
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Wisconsin-Madison, which also approved the protocol used (protocol numbers V00806). The facilities where this research was conducted are fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.

Ferret Vaccine-Challenge Experiment
Five-month-old female ferrets (Triple F Farms), which were serologically negative by hemagglutination inhibition assay for currently circulating human influenza viruses, were used in this study. Six ferrets per group were vaccinated with 15 μg of HA of inactivated whole HK125- Twenty-eight days after the second immunization, ferrets were intranasally challenged with 10 6 PFUs (PFU) of highly pathogenic H7N9 rGD/3-NA294R virus (a neuraminidase inhibitor-sensitive subpopulation of highly pathogenic A/Guangdong/17SF003/2016 H7N9 virus) (4). Clinical signs, bodyweight, and body temperature were monitored daily for 14 days.
Throat and nasal swabs were collected every day until day 7 post-challenge. On day 4 postchallenge, three ferrets from each group were euthanized and organs (lung, trachea, nasal turbinates, olfactory bulbs, and brain tissues pooled from anterior and posterior brain sections) were collected for virus titration.

Hemagglutination Inhibition (HI) Assay
To detect hemagglutination inhibition (HI) activity (https://www.cdc.gov/flu/professionals/laboratory/antigenic.htm), serum samples were treated with receptor-destroying enzyme (RDE; Denka Seiken Co., Ltd) at 37°C for 16-20 h, followed by RDE inactivation at 56°C for 30-60 min. The treated sera were serially diluted 2-fold with PBS in 96-well U-bottom microtiter plates (Thermo Scientific, Rochester, New York, USA) and mixed with the amount of virus equivalent to eight hemagglutination units, followed by incubation at room temperature (25°C) for 30 min. After 50 μL of 0.5% turkey red blood cells was added to the mixtures, they were gently mixed and incubated at room temperature for a further 45 min. HI titers are expressed as the inverse of the highest antibody dilution that inhibited hemagglutination.

Statistical Analysis
Body temperature, bodyweight, nasal, and throat swabs were analyzed using a linear mixed model, with the groups and time as fixed effects, and the animals as random effects.
The commands lmer, lsmeans, and cld were used for the analysis, and all groups were compared to each other (pairwise). The p-values were adjusted using Holm's method. For the comparison of the HI titers, we used two-tailed unpaired t-tests, and adjusted the p-values using Holm's method. The virus titers from the organs were analyzed using one-way ANOVA, followed by Tukey's post-hoc test.

Biosafety and Biosecurity
All recombinant DNA protocols were approved by the University of Wisconsin-Madison's Institutional Biosafety Committee after risk assessments were conducted by the Office of Biologic Safety. In addition, the University of Wisconsin-Madison Biosecurity Task Force regularly reviews the research program and ongoing activities of the laboratory. The task force has a diverse skill set and provides support in the areas of biosafety, facilities, compliance,  Technical Appendix Table 2. Statistical analysis of HI titers of groups 1 and 2 against HK125-HYPR8 in Figure 1,  Technical Appendix Table 3. Statistical analysis of HI titers of groups 1 and 2 against rGD/3-NA294R in Figure 1,  Technical Appendix Table 9. Statistical analyses of nasal swab titers in Figure 2,  Technical Appendix Table 10. Statistical analyses of throat swab titers in Figure 2,  Technical Appendix Table 11. Statistical analyses of throat swab titers in Figure 2,  Technical Appendix Table 12. Statistical analyses of brain titers in Figure 2, Table 13. Statistical analyses of brain titers in Figure 2,