Figure 1.
SDS-PAGE of IgG and F(ab')2 preparations.
Protein samples in SDS buffer were subjected to electrophoresis under non-reducing and reducing conditions through a 4–20% Tris-HEPES SDS-PAGE gel. The gel was stained with Coomassie blue G-250. Molecular mass markers (M) are indicated in kilodaltons. NI, non-immune. The minor band in the non-immune IgG preparation run under non-reducing conditions corresponds to IgG dimers which can sometimes form after freeze-drying. The results are typical of profiles from three separate SDS-PAGE analyses of these particular preparations and of many other analyses of colostrum-derived antibodies .
Table 1.
Hemagglutination inhibition and virus neutralization titers of Ab preparations.
Figure 2.
Virus loads are reduced in the nasal turbinates after treatment with the anti-PR8 IgG preparation.
Unanesthetised BALB/c mice (n = 4) were given 500 pfu of PR8 virus in 10 µl by the intranasal route to establish a localised URT infection. Twenty four hours post-infection, mice were treated with 200 µg, 150 µg, 100 µg or 50 µg of (A) anti-PR8 IgG or non-immune IgG or (B) anti-PR8 F(ab')2 or non-immune F(ab')2 or PBS via the URT route. Five days post-infection, mice were killed and nasal turbinates were collected in 1 ml media. Viral titers were determined by plaque assay on MDCK cell monolayers. The experiment was performed once with 4 mice per group, the organs assayed in quadruplicate and titers averaged to provide a value for that animal. Each symbol represents an individual mouse and the line represents the mean of each group. The bracket with an asterisk indicates a statistically significant difference between the indicated groups.
Figure 3.
Virus load reduction is not enhanced by a second dosing with Ab.
BALB/c mice were given 500 pfu of PR8 virus in 10 µl by the intranasal route to establish a localised URT infection. One and 3 days post-infection, mice were treated with 200 µg of each Ab preparation or PBS via the URT route. Five days post-infection, mice were killed and nasal turbinates were collected in 1 mL media. Viral titers were determined by plaque assay on MDCK cell monolayers. The experiment was performed once with 4 mice per group, the organs assayed in quadruplicate and titers averaged to provide a value for that animal. Each symbol represents an individual mouse and the line represents the mean of each group. Brackets with an asterisk indicate a statistically significant difference between the indicated groups.
Figure 4.
Virus loads are reduced in the lungs after treatment with the anti-PR8 IgG and F(ab')2 preparations.
BALB/c mice (n = 4) were infected with 50 pfu of PR8 virus in 50 µl via the TRT route under penthrane anaesthesia. Twenty four hours post-infection, mice were treated with 1000 µg, 800 µg, 500 µg, 200 µg or 100 µg of (A) anti-PR8 IgG or non-immune IgG or (B) anti-PR8 F(ab')2 or non-immune F(ab')2 or PBS via TRT route. Five days post-infection, mice were killed and lungs were collected in 1 ml media. (C) Mice were infected and treated as in (B) and lungs sampled 7 days post infection. Viral titres were determined by plaque assay on MDCK cell monolayers. The experiment was carried out in its entirety once and results are consistent with repeat experiments on selected doses. Each symbol represents an individual mouse and the line represents the mean of each group. Brackets with an asterisk indicate a statistically significant difference (p<0.001) between the indicated groups.
Figure 5.
Virus loads are reduced but by a lesser extent if Ab treatment is delayed.
BALB/c mice were infected with 50 pfu of PR8 virus in 50 µl via the TRT route under penthrane anaesthesia. Two days post-infection, mice were treated with 1 mg of the Ab preparations or PBS via the TRT route. Five days post-infection, mice were killed and lungs were collected in 1 ml media. Viral titres were determined by plaque assay on MDCK cell monolayers. The experiment was performed once with 5 mice per group, the organs assayed in quadruplicate and titers averaged to provide a value for that animal. Each symbol represents an individual mouse and the line represents the mean of each group. Brackets with an asterisk indicate a statistically significant difference between the indicated groups.
Figure 6.
Anti-PR8 IgG and F(ab')2 preparations can completely prevent influenza infection.
BALB/c mice (n = 5) were given 1 mg of the Ab preparations or PBS via the TRT route in 50 µl under penthrane anaesthesia. One, 2, 3 or 7 days later, mice were infected with 50 pfu of PR8 virus in 50 µl via the TRT route under penthrane anaesthesia. One day post-infection, mice were killed and lungs were collected in 1 ml media. Viral titres were determined by plaque assay on MDCK cell monolayers. Each symbol represents an individual mouse and the line represents the mean of each group. The experiment was carried out in its entirety once and results are consistent with repeat experiments on selected doses.
Figure 7.
Anti-PR8 IgG and F(ab')2 preparations can prevent severe weight loss and death from a lethal dose of PR8 virus.
BALB/c mice (n = 5) were treated with 1 mg of the Ab preparations or PBS in 50 µl i.n. under penthrane anaesthesia. On days 3 (A and C) or 7 (B and D) after Ab administration, mice were infected with 500 pfu of PR8 virus in 50 µl via the TRT route under penthrane anaesthesia. Mice were monitored daily for 16 days and killed at the humane endpoint. The experiment was performed once in accordance with animal ethics approvals. The change in percentage of the starting weight (A and B) and Kaplan-Meier survival curves (C and D) are shown.
Figure 8.
Anti-PR8 IgG and F(ab')2 preparations can prevent severe weight loss and death from an established lethal PR8 infection.
BALB/c mice (n = 4) were infected with a lethal dose (500 pfu in 50 µl) of PR8 virus via the TRT under penthrane anaesthesia. One day post-infection, mice were treated with 1 mg of the Ab preparations or PBS in 50 µl via the TRT route. Mice were monitored daily for 16 days and killed at the humane endpoint. The experiment was performed once in accordance with animal ethics approvals. The change in percentage of the starting weight (A) and Kaplan-Meier survival curves (B) are shown.
Figure 9.
Half-lives of intranasally-delivered bovine IgG in the nose, lungs and serum.
The bovine anti-PR8 IgG preparation (1 mg) was administered in a volume of 50 µl by the i.n. route to anesthetized mice. At 1, 12, 24 and 36 hr post-administration, mice were killed and bovine IgG was detected by capture ELISA in nasal turbinate and lung homogenates and in sera. The total amount of Ab was determined by reference to a standard curve. Linear regression analysis was used to calculate the half-lives of bovine IgG in the different sites. The data are representative of two experiments.