Volume 24, Issue 5, Pages 991-1002 (May 2016) H7N9 Live Attenuated Influenza Vaccine Is Highly Immunogenic, Prevents Virus Replication, and Protects Against Severe Bronchopneumonia in Ferrets  Jørgen de Jonge, Irina Isakova-Sivak, Harry van Dijken, Sanne Spijkers, Justin Mouthaan, Rineke de Jong, Tatiana Smolonogina, Paul Roholl, Larisa Rudenko  Molecular Therapy  Volume 24, Issue 5, Pages 991-1002 (May 2016) DOI: 10.1038/mt.2016.23 Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Mutations as a result of egg adaptation. Sequencing diagrams of HA (nt 421 and 499) and NA (nt 29) of WT A/Anhui/1/2013 (H7N9) influenza after multiple passaging on eggs and a single passage on MDCKs. Molecular Therapy 2016 24, 991-1002DOI: (10.1038/mt.2016.23) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Virus replication in the respiratory tract and brain. (a) Virus titers in the nasal turbinates, trachea, lung, olfactory bulb (OB), and brain of ferrets sacrificed 4 days after intranasal infection with either WT H7N9 (red triangles), H2N2 master donor virus (MDV; blue circles), or H7N9 live attenuated influenza vaccine (LAIV) (black squares). No virus replication was detected in the intestines or the spleen (not shown). (b) Nose and (c) throat swabs were performed on all animals (six per group) 1 day prior to challenge and 2 and 4 days after infection. After termination of three animals per group 4 days post infection (d.p.i.), sampling was continued in the remaining three animals on days 6, 8, and 10. The virus titers in the homogenized tissue samples or transport buffer of the swabs were determined by end-point titration on Madin–Darby canine kidney cells using a fivefold serial dilution. WT H7N9 titrations were incubated at 37 °C whereas H2N2 MD and H7N9 LAIV titrations were performed at 32 °C. Presented are the individual 10 log transformed titers connected with a line through the averages (b and c only). Dotted horizontal lines indicate limit of detection. Dotted vertical lines indicate day of termination of first three ferrets. Molecular Therapy 2016 24, 991-1002DOI: (10.1038/mt.2016.23) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Functional and neutralizing antibody responses. (a) Hemagglutination inhibition, (b) neuraminidase inhibition, and (c) virus neutralization antibody titers detected in sera during the course of the study in serum of placebo (black squares), 1×LAIV (blue circles), or 2×LAIV (red triangles) intranasally vaccinated ferrets. (d) Cross-reactive hemagglutination inhibition titers at the day before challenge. Titers were measured against four hemagglutinating units of H7N9 LAIV as a representative of the hemagglutinin of (a) WT virus, (d) WT H7N9, H7N3, or H7N7 using a concentration of 1% horse erythrocytes (a), an H6N9 (RN19/13-human reassortant strain) dilution with a neuraminidase activity resulting in ∼OD 1 after incubation of 1 hour at 37 °C (b), and against 100 TCID50 of WT H7N9 (c). Inhibition of neuraminidase was considered at an OD of less than three times the SD below the maximal OD (standard H6N9). VN titer was determined at 50% virus neutralization according to Reed and Muench.46 Presented are the individual titers and a line connecting the averages. Vertical dotted lines indicate the following: a. 1st vaccination 2×LAIV; b. 1st vaccination 1×LAIV; c. 2nd vaccination 2×LAIV; d. challenge. LAIV, live attenuated influenza vaccine. Molecular Therapy 2016 24, 991-1002DOI: (10.1038/mt.2016.23) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Fever and weight loss. Body temperature was recorded every 30 minutes by transponders that were placed in the intraperitoneal cavity: (a.1) placebo; (a.2) 1×LAIV; (a.3) 2×LAIV. To analyze fever development, the temperature difference compared with baseline was calculated and the average ΔT per group was plotted. The dotted lines indicate the SD. The several sharp decreases in temperature are a result of sedation indicated by vertical dotted lines: a. baseline swabs and weight; b. challenge; c. swabs and weight; d. section. The temperature decrease of animals that succumbed to the infection started a couple of hours prior to death, and therefore in the placebo group five animals (one transponder had a power down) were included until day 39 (2.00 p.m.); four animals until day 40 (8.00 a.m.), and the remaining two animals until section. (b) Body weight prior to challenge was set at 100%, and percentage weight loss was subtracted from baseline. Presented are the individual body weight loss and lines connecting the averages. Placebo (black squares), 1×LAIV (blue circles) or 2×LAIV–vaccinated (red triangles)–vaccinated ferrets.##P < 0.01(1×LAIV,) $P < 0.05 (2×LAIV), and $$P < 0.01(2×LAIV) indicate difference compared with placebo by the Mann–Whitney test. LAIV, live attenuated influenza vaccine. Molecular Therapy 2016 24, 991-1002DOI: (10.1038/mt.2016.23) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Virus replication in the respiratory tract. Virus titers in the throat 2 and 3 days post challenge (d.p.c.) and in the trachea and lung 3 d.p.c. in placebo (black squares), 1×LAIV–vaccinated (blue circles), or 2×LAIV (red triangles)–vaccinated ferrets. The virus titers in the transport buffer of the swabs or homogenized tissue samples were determined by end-point titration on Madin–Darby canine kidney cells using a fivefold serial dilution. Presented are the individual 10 log transformed titers, their averages (bar), and SD (error bar). Dotted horizontal lines indicate limit of detection. Throat swabs taken prior to challenge were negative for virus replication (data not shown). LAIV, live attenuated influenza vaccine. Molecular Therapy 2016 24, 991-1002DOI: (10.1038/mt.2016.23) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Affected lung tissue at the macroscopic level and edema formation. Representative images of inflated lungs (dorsal view) of (a.1) placebo, (a.2) 1×LAIV–vaccinated, and (a.3) 2×LAIV–vaccinated ferrets. Bar = 2 cm. (b) During section, the lung lobes were macroscopically examined and an estimate of the percentage tissue that was affected (dark red) was recorded. (c) The relative lung weight (RLW) was calculated as the percentage of the lung weight 3 days post challenge (d.p.c.) relative to the body weight at the day of challenge. An increase of the RLW is caused by edema formation and is a measure for pneumonia. Presented are the individual values, their means (bar), and SDs (error bar). LAIV, live attenuated influenza vaccine. Molecular Therapy 2016 24, 991-1002DOI: (10.1038/mt.2016.23) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Microscopic analysis of lung pathology. Representative hematoxylin and eosin (HE) stained lung sections of (a.1) placebo, (a.2) 1×LAIV–vaccinated, and (a.3) 2×LAIV–vaccinated ferrets. Panel (a.1) shows necrotizing and denudation of inflamed bronchioli with luminal plugs of inflammatory exudate (indicated by *), accompanied by a small peribronchiolar rim of lymphocytes and some polymorphonuclear (PMN) cells. Hyperemia is strongly present, and the alveolar space is partly filled with edema, fibrin, and debris; PMN cells are regularly present (indicated by ♦). Panels (a.2) and (a.3) show peribronchiolitis and (a.2) also an enhanced cellularity of the interstitium. Alveolar inflammation is minimal or absent. Single-vaccinated ferrets (a.2) reveal a moderate hyperplasia of the bronchiolar epithelial lining, some luminal exudate of PMN cells, and a strong peribronchiolar accumulation of lymphocytes. Double-vaccinated animals (a.3) present a slight peribronchiolar infiltrate and slight hyperplasia of the epithelial lining and a minimal luminal exudate. Bar = 100 µm. (b) The end score is an overall pathology score which is determined based on the amount of tissue affected, the degree of inflammation, and the amount of damage in the left caudal and cranial lung lobe. The scores range from 0 (no aberrations) to 5 (severely affected). (c) The estimated percentage of lung parenchyma involved in the infection pathology. Bars represent the mean and the error bars represent the SD. Statistical analysis was performed using (b) an adapted version of the Mann–Whitney test for ordinal data with a small sample size and (c) the classical Mann–Whitney test. LAIV, live attenuated influenza vaccine. Molecular Therapy 2016 24, 991-1002DOI: (10.1038/mt.2016.23) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Detailed histopathological analysis. Pathology parameters were categorized according to their relation to damage (upper panel), acute inflammation (middle panel), and lymphocytic infiltration (lower panel), and these were assessed for the different lung compartments: bronchioli, blood vessels, interstitium, and alveoli. For each category, a final score was determined ranging from 0 (no aberrations) to 5 (severely affected). The category damage includes directly virus-induced effects, the category acute inflammation includes innate effects, and the category lymphocytic infiltrate includes mostly adaptive immune effects. The bronchi only showed minimal effects (not presented). Statistical analysis was performed using an adapted version of the Mann–Whitney test for ordinal data with a small sample size. Molecular Therapy 2016 24, 991-1002DOI: (10.1038/mt.2016.23) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions