A mouse model for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans

Abstract

During 1997 in Hong Kong, 18 human cases of respiratory illness, including 6 fatalities, were caused by highly pathogenic avian influenza A (H5N1) viruses. Since H5 viruses had previously been isolated only from avian species, the outbreak raised questions about the ability of these viruses to cause severe disease and death in humans. To better understand the pathogenesis and immunity to these viruses, we have used the BALB/c mouse model. Four H5N1 viruses replicated equally well in the lungs of mice without prior adaptation but differed in lethality for mice. H5N1 viruses that were highly lethal for mice were detected in multiple organs, including the brain. This is the first demonstration of an influenza A virus that replicates systemically in a mammalian species and is neurotropic without prior adaptation. The mouse model was also used to evaluate a strategy of vaccination against the highly pathogenic avian H5N1 viruses, using an inactivated vaccine prepared from nonpathogenic A/Duck/Singapore-Q/F119-3/97 (H5N3) virus that was antigenically related to the human H5N1 viruses. Mice administered vaccine intramuscularly, with or without alum, were completely protected from lethal challenge with H5N1 virus. Protection from infection was also observed in 70% of animals administered vaccine alone and 100% of mice administered vaccine with alum. The protective effect of vaccination correlated with the level of virus-specific serum antibody. These results suggests a strategy of vaccine preparedness for rapid intervention in future influenza pandemics that uses antigenically related nonpathogenic viruses as vaccine candidates.

FIG. 1

Comparison of lethality of human H5N1 influenza viruses for BALB/c mice. Groups of 10 mice were infected i.n. with 10⁴ (A), 10³ (B), or 10² (C) MID₅₀ of HK/483 (●), HK/485 (○), HK/156 (▾), or HK/486 (▿) virus and examined daily for 14 days.

FIG. 2

Replication of influenza A (H5N1) viruses in mice. Mice were infected with 100 MID₅₀ of each virus; tissues and blood were collected on days 4 (■) and 6 (□) p.i., and titrated in eggs. The mean virus titers from three mice per group are shown. The limit of virus detection was 10^1.2 EID₅₀/ml for lungs and 10^0.8 EID₅₀/ml for blood and other tissues. ∗ and †, P < 0.01 and P < 0.05, respectively, versus groups infected with HK/156 or HK/486 virus.

FIG. 3

Immunostaining of viral antigens in lung and brain from mice infected with 1,000 MID₅₀ of HK/483 virus. (A) Immunostaining in bronchial epithelium and subepithelial tissue from a mouse that succumbed to infection on day 7 p.i. (B) Higher magnification of a bronchus showing mainly nuclear, and to a less extent cytoplasmic, staining of bronchial epithelial cells. Note immunostaining in association with necrotic detached epithelial cells in bronchial lumen. (C) Brain collected on day 6 p.i. showing a focus of antigen-positive cells. (D) High-power magnification showing nuclear and cytoplasmic immunostaining of glial cells and neurons (naphthol-fast red with hematoxylin counterstain; original magnifications, ×50 [A], ×158 [B], ×50 [C], and ×158 [D]).

FIG. 3

Immunostaining of viral antigens in lung and brain from mice infected with 1,000 MID₅₀ of HK/483 virus. (A) Immunostaining in bronchial epithelium and subepithelial tissue from a mouse that succumbed to infection on day 7 p.i. (B) Higher magnification of a bronchus showing mainly nuclear, and to a less extent cytoplasmic, staining of bronchial epithelial cells. Note immunostaining in association with necrotic detached epithelial cells in bronchial lumen. (C) Brain collected on day 6 p.i. showing a focus of antigen-positive cells. (D) High-power magnification showing nuclear and cytoplasmic immunostaining of glial cells and neurons (naphthol-fast red with hematoxylin counterstain; original magnifications, ×50 [A], ×158 [B], ×50 [C], and ×158 [D]).

FIG. 4

Serum HI antibody responses following one or two doses of dk/Sing vaccine. Mice were vaccinated i.m. with alum alone (A), 10 μg of dk/Sing vaccine alone (B), or vaccine with alum (C). Sera from 17 to 20 mice per group were collected 3 weeks after the first and second vaccinations and tested individually for HI antibody against HK/156 virus. HI titers are expressed as a log₂ value of the reciprocal of the highest dilution of serum inhibiting agglutination of 0.5% of turkey erythrocytes at 4 HA units of virus. Antibody responses shown in panel C were significantly greater than the corresponding responses shown in panel B (P < 0.001). A log₂ value of ≥5.3 represents an HI titer of ≥40. Solid bars represent the GMT.