Vaccine efficacy in nonhuman primates assessed on the basis of lung lesions.

<p>Eight macaques were euthanized on day 3 postchallenge with AH/05 virus (A–D) or BHG/05 virus (E–H). Vaccinated animals (A,B,E,F) had less extensive bronchopneumonia (i.e., smaller foci of consolidation) than did unvaccinated animals (C,D,G,H). The vaccinated animals also showed prominent peribronchial lymph follicles (a, e; arrows), and their consolidated lung areas lacked viral antigen-positive cells (B,F). By contrast, the unvaccinated animals had lung lesions of moderate size with a wide consolidated area (C,G; outlined by yellow dashes), smaller and less abundant peribronchial lymph follicles (C,G; arrows), and pneumonic lesions containing many antigen-positive cells (D,H; brown pigment). (I) Schematic diagrams indicating distribution of pathologic lesions in the lungs of animals vaccinated and challenged with AH/05 (V1 and V2); nonvaccinated and challenged with AH/05 (C1 and C2); vaccinated and challenged with BHG/05 (V3 and V4); and nonvaccinated and challenged with BHG/05 (C3 and C4). In vaccinated animals, scant-to-moderate bronchopneumonia was present in each lobe, but viral antigens were not detected in the lesions (V1, V2, V3, and V4; purple). By contrast, more severe bronchopneumonia was observed in nonvaccinated macaque lungs (C1, C2, C3, and C4). Moreover, viral antigens were prominent in the pneumonic lesions in the most affected lung lobes (C1, C2, C3, and C4; red). One lung lobe was entirely affected by pneumonia after infection with the BHG/05 virus (C3). Purple, bronchopneumonia without viral antigen; red, bronchopneumonia with viral antigen.</p

Antibody responses of nonhuman primates.

<p>(A) H5N1-specific antibody levels assessed by ELISA. Hemagglutination-inhibition (HI) antibody to AH/05 (B) and BHG/05 (C) with chicken erythrocytes, and microneutralization (NT) antibody to the AH/05 (D) and BHG/05 (E) viruses. The HI antibody titers with horse erythrocytes were 4- to 8-fold higher than those with chicken erythrocytes (data not shown). Titers are reported for individual vaccinated animals. Blue dashed lines indicate the lower limit of detection. Wpd1/2, week postvaccination dose 1 or 2; wpc, week postchallenge. The P values indicate the antibody titers with a significant increase from the preceding time point. In the control animals, the HI and NT antibodies at all time points are the same as the pretested values, only the ELISA antibody titers, ranged 400–800, were detected at two weeks postchallenge (data not shown).</p

Pathological lesions and antigen distribution in tissues of nonhuman primates infected with H5N1 viruses.

<p>Animals were vaccinated twice (4-week interval) with AH/AA<i>ca</i> and challenged with AH/05 or BHG/05 virus. Tissues for pathological examination were collected 3 days after viral challenge. Tra, trachea; Bro, Bronchus; TBLN, tracheobronchial lymph node.</p>b<p>Pathological lesions/viral antigens. −, no pathological change/antigen. +, limited pathological change/antigen. ++, moderate pathological change/antigen. +++, severe pathological change/abundant antigen.</p

Viral replication in nonhuman primates.

<p>Virus titers were determined in embryonated eggs injected with tissue homogenates on day 3 (A,B) and day 15 (C) postchallenge with AH/05 virus or BHG/05 virus. Virus was not detected in tissues harvested day 15 postchallenge from animals challenged with the BHG/05 virus (data not shown). Titers are reported for tissues from individual animals, as log₁₀ EID₅₀/g tissue. The dashed blue lines indicate the lower limit of detection. (D) Nasal swabs were collected from all living animals on days 2, 4, and 6 postchallenge for virus isolation in eggs.</p

Vaccine efficacy of the AH/AA<i>ca</i> virus in mice.

<p>(A) HI and (B) NT antibody responses to homologous (AH/05) and heterologous (BHG/05) viruses after intranasal vaccination with 10⁶ EID₅₀ of AH/AA<i>ca</i> in a 50-µl volume. Serum samples were collected on day 0 prevaccination (blue), 4 weeks after the first (orange) and 4 weeks after the second vaccination (pink). Asterisks indicate a statistically significant difference from antibody titers measured at the preceding time point: **, P<0.01, *, P<0.05. (C–H) Protective efficacy against challenge with the AH/05 (C–E) or BHG/05 virus (F–H). Weight changes (D and G) and survival rates (E and H) are shown only for the groups that were immunized once. The data in panels A–C and F are reported as means±s.d.; the dashed blue lines in these panels indicate the lower limit of detection. p.c., postchallenge.</p

Body temperature of nonhuman primates after challenge with AH/05 virus.

<p>Change in body temperature in nonhuman primates after challenge with AH/05 virus (A) or BHG/05 virus (B). Changes were calculated by subtracting the mean temperature 3 days before challenge from the temperature recorded on the indicated day.</p

Replication of the H5N1 <i>ca</i> reassortants and the wild-type H5N1 viruses in mice.

a<p>Six-week-old BALB/c mice (3 per group), inoculated intranasally with 10⁶ EID₅₀ of the indicated virus in a 50-µl volume, were killed on day 3 postinoculation and their organs were collected for virus titration in eggs. <, no virus was isolated from that sample.</p>b<p>P value was<0.01 compared with the titers in the corresponding organs of the AH/05-inoculated mice.</p

T cell peptides and their corresponding T cell subsets against H5 HA in monkeys vaccinated with AH/AA<i>ca</i>.

a<p>Sequences were based on the HA gene of A/Anhui/1/05 virus.</p>b<p>The numbers of IFN-γ–positive T lymphocytes in PBMC samples expressed as spot-forming cells (SFC)/million PBMCs were determined by HA overlapping peptides and IFN–ELISPOT assay.</p>c<p>T cell subset of the HA-specific T cells in PBMC samples was further determined by cell depletion using magnetic beads against CD8 and IFN-γ ELISPOT assay.</p

Attenuation phenotype of the recombinant AH/AA<i>ca</i> virus in chickens.

a<p>Six-week-old specific-pathogen free chickens were inoculated i.n. with 10⁶ EID₅₀ of virus in a 0.1-ml volume. Swabs were collected from the birds on day 3 and day 5 postinoculation and titrated in eggs. <, virus not detected. data are means±standard deviations. No. sick/deaths/total; numbers of chickens that were sick and died, as well as the total number of chickens during the observation period. Birds that showed disease signs, such as depression and ruffled feathers, but recovered at the end of the observation were counted as sick animals. SC/total, number of chickens that seroconverted out of the total number of chickens at the end of the 2-week observation period. NA, all birds died by the end of the observation period and thus could not be studied for sera conversion.</p>b<p>Six-week-old white Leghorn chickens housed in high-efficiency particulate air-filtered isolators were inoculated i.v. with 0.2 ml of a 1∶10 dilution of bacterium-free allantoic fluid containing virus for intravenous pathogenicity index (IVPI) testing, based on recommendations of the Office International Des Epizooties.</p>c<p>Only animals that showed disease signs but recovered by the end of the observation period were identified as sick birds.</p

Antibody response induced by the H5N1 <i>ca</i> reassortant viruses in mice.

<p>Group of five six-week-old BALB/c mice were inoculated with two dose, in a 4 week interval, of 10⁶ EID₅₀ of the indicated H5N1 <i>ca</i> reassortant virus. Four weeks after dose 1 or dose 2, sera were collected for determining the HI and NT antibodies using the homologous wild type H5N1virus.</p