Article Figures & Data
Figures
- 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 101.2 EID50/ml for lungs and 100.8 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. 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.
Tables
- Table 1.
Characteristics of influenza A H5N1 viruses
Virus Groupa Case description Passage no. Infectivity titer (log10/ml)b MID50c LD50d Age (yr)/gender Outcome MDCK Eggs TCID50 EID50 HK/483 B 13/female Fatal 1 3 8.5 9.0 2.2 2.4 HK/485 B 24/female Severe/recovered 2 2 8.5 8.7 1.1 2.9 HK/156 A 3/male Fatal 0 3 7.7 10.0 3.2 5.9 HD/486 A 5/female Mild/recovered 2 4 8.7 9.0 1.2 >7 X-31 (H3N2) NAe NA 0 >10 7.3 8.5 0.7 >5.2 ↵a Antigenic/genetic group of H5N1 viruses isolated from humans (3).
↵b Calculated by the method of Reed and Muench (24) from the results of serial titrations of viruses in MDCK cells and embryonated eggs.
↵c Expressed as the EID50 required to give 1 MID50.
↵d Expressed as the EID50 required to give 1 LD50.
↵e NA, not applicable.
- Table 2.
HI reactivity of serum samples from vaccinated mice for influenza A H5 viruses
Serum HI antibody titer dk/Sing (H5N3) H5N1 group A H5N1 group B HK/156 HK/486 HK/516 HK/538 HK/483 HK/485 HK/491 HK/514 Vaccinated micea dk/Sing 80 80 40 40 40 40 40 80 40 dk/Sing + alum 320 320 160 320 640 160 80 320 160 Alum 10 10 10 10 10 10 10 10 10 Infected ferretsb dk/Sing 160 160 80 160 160 40 40 80 80 HK/156 160 160 160 160 160 20 20 80 80 HK/483 160 160 80 160 160 160 40 80 80 ↵a Serum samples from groups of 17 to 22 mice immunized with dk/Sing vaccine with or without alum or alum alone were collected 3 weeks after the second vaccination and were pooled for HI assay against H5N1 group A and group B viruses.
↵b Serum samples from single ferrets infected with dk/Sing, HK/483, or HK/156 virus were concentrated fourfold. The ferret infected with HK/156 virus was also inoculated i.n. with A/Turkey/Wisconsin/68 virus (H5N9).
- Table 3.
Protective efficacy of dk/Sing (H5N3) vaccine against lethal infection with HK/483 virus
Vaccine group Prechallenge antibody titera against: Mean virus titer (log10 EID50/ml) ± SDb No. protected/total no.c HK/156 HK/483 HI Neutralization HI Neutralization Lung Brain Infection Death dk/Singd 80 400 40 100 2.6 ± 0.6e ≤0.8e 7/10f 12/12e dk/Sing + alum 1,280 8,000 320 1,600 ≤1.2e ≤0.8e 8/8e 12/12e Alum 10 25 10 25 6.3 ± 0.9 2.5 ± 1.0 0/7 0/10 ↵a Serum samples pooled from 10 mice per group were used.
↵b Mean virus titers and protection from infection were determined on day 6 p.i. Titers represent means ± SD of 7 to 10 mice per group.
↵c Mice were monitored for 14 days after challenge. Mice in the alum-vaccinated group died on day 6 or 7.
↵d Mice were vaccinated with two doses of 10 μg of formalin-inactivated dk/Sing vaccine and challenged 4 months later with 50 LD50 of HK/483 virus.
↵e P < 0.001 compared with alum-vaccinated group.
↵f P < 0.01 compared with alum-vaccinated group.
