Prophylactic and therapeutic efficacy of human monoclonal antibodies against H5N1 influenza

Abstract

Background: New prophylactic and therapeutic strategies to combat human infections with highly pathogenic avian influenza (HPAI) H5N1 viruses are needed. We generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs) and tested their efficacy for prophylaxis and therapy in a murine model of infection.

Methods and findings: Using Epstein-Barr virus we immortalized memory B cells from Vietnamese adults who had recovered from infections with HPAI H5N1 viruses. Supernatants from B cell lines were screened in a virus neutralization assay. B cell lines secreting neutralizing antibodies were cloned and the mAbs purified. The cross-reactivity of these antibodies for different strains of H5N1 was tested in vitro by neutralization assays, and their prophylactic and therapeutic efficacy in vivo was tested in mice. In vitro, mAbs FLA3.14 and FLD20.19 neutralized both Clade I and Clade II H5N1 viruses, whilst FLA5.10 and FLD21.140 neutralized Clade I viruses only. In vivo, FLA3.14 and FLA5.10 conferred protection from lethality in mice challenged with A/Vietnam/1203/04 (H5N1) in a dose-dependent manner. mAb prophylaxis provided a statistically significant reduction in pulmonary virus titer, reduced associated inflammation in the lungs, and restricted extrapulmonary dissemination of the virus. Therapeutic doses of FLA3.14, FLA5.10, FLD20.19, and FLD21.140 provided robust protection from lethality at least up to 72 h postinfection with A/Vietnam/1203/04 (H5N1). mAbs FLA3.14, FLD21.140 and FLD20.19, but not FLA5.10, were also therapeutically active in vivo against the Clade II virus A/Indonesia/5/2005 (H5N1).

Conclusions: These studies provide proof of concept that fully human mAbs with neutralizing activity can be rapidly generated from the peripheral blood of convalescent patients and that these mAbs are effective for the prevention and treatment of H5N1 infection in a mouse model. A panel of neutralizing, cross-reactive mAbs might be useful for prophylaxis or adjunctive treatment of human cases of H5N1 influenza.

Figure 1. Passive Immunization and Survival after Challenge with A/Vietnam/1203/04 (H5N1)

BALB/c mice (n = 5 per group) were passively immunized by i.p. injection of graded doses of anti-H5N1 mAbs FLA3.14 or FLA5.10, the control mAbs D2.2 or A146, or hyperimmune sheep antisera specific for the H5N1 HA protein. Mice were challenged i.n. with 50 μl of A/Vietnam/1203/04 (10⁵ TCID₅₀/mouse) 24 h later. The data show the Kaplan-Meier survival curves for the 2 wk period of observation. Mice that received either sheep anti-H5 antisera or FLA5.10 were completely protected from lethal infection (sheep antisera or FLA5.10 versus D2.2, p = 0.001, log rank test). FLA3.14 afforded significant protection at 20 mg/kg and 10 mg/kg (FLA3.14 versus D2.2, p = 0.001, log rank test). The lowest dose of FLA3.14 (1 mg/kg) delayed time to death (FLA3.14 versus D2.2, p = 0.02, log rank test), but did not prevent fatal infection.

Figure 2. Pulmonary Virus Titers and Extrapulmonary Dissemination of A/Vienam/1203/04 (H5N1) Following Passive Immunization

The data depict the mean (± interquartile range) viral titer in (A) lungs, (B) brain, and (C) spleen of groups of five BALB/c mice passively immunized by i.p. injection of mAbs FLA3.14, FLA5.10, or the control mAb D2.2, then challenged i.n. with 50 μl of A/Vietnam/1203/04 (10⁵ TCID₅₀/mouse) 24 h later. On days 2 and 4 there was significantly less virus recovered in splenic and pulmonary tissue of mice that had received either FLA3.14 or FLA5.10 than mice that had received the control mAb, D2.2. (* p < 0.01 versus D2.2; ** p < 0.001 versus D2.2). The lower limit of detection (1.5 log₁₀ TCID₅₀/g) is shown by the arrow.

Figure 3. Histopathology in Pulmonary Tissue of Passively Immunized and Challenged Mice

(A) Hematoxylin and eosin-stained lung sections (40×) revealed diffuse interstitial pneumonia (I) and bronchial and bronchiolar (Br) involvement in a mouse infected with influenza A/VN/1203/04 (H5N1) after i.p. injection of the control mAb D2.2. (B) Mouse given mAb FLA5.10 prior to infection with influenza A/VN/1203/04 (H5N1), showing much less lung involvement than in (A). (C) Immunohistochemistry (40×) revealed H5 antigen in bronchi, bronchioles (Br), and interstitial lesions (I) in a mouse given influenza A/VN/1203/04 (H5N1) following i.p. injection of the control mAb D2.2. (D) H5 antigen in bronchus (Br) and not in bronchioles or interstitial areas (I) in a mouse given mAb FLA5.10 prior to influenza challenge. (E) High magnification (100×) of (C) showing abundant H5 antigen in interstitial alveolar lesions (I) and bronchiolar epithelium (Br). (F) High magnification of (D) (100×) showing H5 antigen only focally in a bronchus (Br) and not in the interstitial alveolar areas (I).

Figure 4. mAb Therapy and Survival in Mice with Established A/Vietnam/1203/04 (H5N1) Infection

The data show the Kaplan-Meier survival curves for groups of BALB/c mice (n = 5 per group) that were infected i.n. with 5 LD₅₀ of A/Vietnam/1203/04, then 24, 48, or 72 h later treated by i.p. injection with the control mAb D2.2, or the anti-H5N1 mAbs FLA3.14, FLA5.10, FLD20.19, or FLD21.140, each at 50 mg/kg body weight. Postinfection therapy at 24, 48, or 72 h with mAbs FLA3.14, FLA5.10, FLD20.19, or FLD21.140 was associated with significant protection from lethal infection at all time points (FLA3.14, FLA5.10, FLD20.19, or FLD21.140 versus D2.2, p = 0.003, log rank test).

Figure 5. mAb Therapy and Survival in Mice with Established A/Indonesia/5/2005 (H5N1) Infection

The data show the Kaplan-Meier survival curves for groups of BALB/c mice (n = 5 per group) that were infected i.n. with 5 LD₅₀ of A/Indonesia/5/2005 (H5N1) then 24 h later treated by i.p. injection with the control mAb D2.2, or the anti-H5N1 mAbs FLA3.14, FLA5.10, FLD20.19, or FLD21.140, each at 50 mg/kg body weight. Postinfection therapy at 24 h with mAbs FLA3.14, FLD20.19, or FLD21.140, but not FLA5.10 or D2.2, was associated with absolute protection from lethal infection (FLA3.14, FLD21.140 or FLD20.19 versus D2.2, p = 0.003, log rank test).