Protection of mice against lethal infection with highly pathogenic H7N7 influenza A virus by using a recombinant low-pathogenicity vaccine strain

Abstract

In 2003, an outbreak of highly pathogenic avian influenza occurred in The Netherlands. The avian H7N7 virus causing the outbreak was also detected in 88 humans suffering from conjunctivitis or mild respiratory symptoms and one person who died of pneumonia and acute respiratory distress syndrome. Here we describe a mouse model for lethal infection with A/Netherlands/219/03 isolated from the fatal case. Because of the zoonotic and pathogenic potential of the H7N7 virus, a candidate vaccine carrying the avian hemagglutinin and neuraminidase proteins produced in the context of the high-throughput vaccine strain A/PR/8/34 was generated by reverse genetics and tested in the mouse model. The hemagglutinin gene of the recombinant vaccine strain was derived from a low-pathogenicity virus obtained prior to the outbreak from a wild mallard. The efficacy of a classical nonadjuvanted subunit vaccine and an immune stimulatory complex-adjuvanted vaccine was compared. Mice receiving the nonadjuvanted vaccine revealed low antibody titers, lack of clinical protection, high virus titers in the lungs, and presence of virus in the spleen, liver, kidneys, and brain. In contrast, mice receiving two doses of the immune stimulatory complex-adjuvanted vaccine revealed high antibody titers, clinical protection, approximately 1,000-fold reduction of virus titers in the lungs, and rare detection of the virus in other organs. This is the first report of an H7 vaccine candidate tested in a mammalian model. The data presented suggest that vaccine candidates based on low-pathogenicity avian influenza A viruses, which can be prepared ahead of pandemic threats, can be efficacious if an effective adjuvant is used.

FIG. 1.

Loss of body weight as the result of infection with influenza virus A/Netherlands/219/03. Mice were infected intranasally with four different infectious doses: 10² (□), 3 × 10³ (○), 10⁵ (▪), and 3 × 10⁶ (•) EID₅₀. Following infection, mice were weighed daily. Percent body weight was calculated compared to body weight at the time of infection.

FIG. 2.

Overview of the vaccination strategies used to test H7N7 vaccine preparations. Six groups of six mice each were vaccinated once or twice intramuscularly with the different vaccine preparations as indicated (↓). On day 4 after challenge, three animals from each group were sacrificed (open cross). On days 6 and 7 after challenge animals from groups I, II, III, and IV were sacrificed due to severity of signs of disease (cross). On day 14 all remaining animals were sacrificed (open crosses). V: vaccination; C: challenge.

FIG. 3.

Serum HI antibody titers against A/Netherlands/219/03 after the first and second vaccinations. Blood samples were taken before the start of the experiment and at 3 weeks after the first and second vaccinations. Serum was used in an HI test against A/Netherlands/219/03. Indicated are geometric mean titers and 95% confidence intervals. The asterisk indicates that in this group three mice had an HI titer below the detection limit and three mice had a titer of 40. ND, not detected. Groups: PBS and ISCOM-measles controls (▪); two 5-μg H7N7 vaccinations (▴); one 5-μg ISCOM-H7N7 vaccination (□); two 1-μg ISCOM-H7N7 vaccinations (○); two 5-μg ISCOM-H7N7 vaccinations (▵).

FIG. 4.

Loss of body weight and survival of vaccinated mice after challenge with influenza virus A/Netherlands/219/03. Vaccinated mice were challenged intranasally with 3 × 10³ EID₅₀ of influenza A virus A/Netherlands/219/03. After challenge, mice were weighed daily. Percent body weight per group was calculated compared to body weight at the time of challenge (A). Mice were sacrificed either due to the severity of their disease signs at day 6 or 7 or at the end of the experiment on day 14 after challenge. The dotted line indicates that one or two mice out of the group were already sacrificed and data are thus based on the body weights of the remaining mice. The percentage of mice surviving the lethal challenge as a function of time is also shown (B). Since three mice out of each group were sacrificed at day 4 after infection, this graph is based on the survival of the remaining three mice from day 5 after infection onwards. Groups: PBS (▪); ISCOM-measles (•); two 5-μg H7N7 vaccinations (▴); one 5-μg ISCOM-H7N7 vaccination (□); two 1-μg ISCOM-H7N7 vaccinations (○); two 5-μg ISCOM-H7N7 vaccinations (▵).

FIG. 5.

Virus titers in mice after challenge with influenza virus A/Netherlands/219/03. Vaccinated mice were challenged intranasally with 3 × 10³ EID₅₀ of influenza A virus A/Netherlands/219/03. On day 4 after challenge three mice from each group were sacrificed, tissues were collected, and virus titers in lungs, spleen, liver, kidney, and brain were determined in MDCK cells. The geometric mean virus titer per group was calculated. To calculate the geometric mean, the cutoff value was used for negatives. Error bars indicate the standard deviation. The dotted line indicates the cutoff value of the assay for each of the organs. Asterisks indicate that only one of the tested animals in the group was positive. Roman numbers refer to the vaccination status as shown in Fig. 2.