Passive immunoprophylaxis and therapy with humanized monoclonal antibody specific for influenza A H5 hemagglutinin in mice

Abstract

Background: Highly pathogenic avian H5N1 influenza virus is a major public health concern. Given the lack of effective vaccine and recent evidence of antiviral drug resistance in some isolates, alternative strategies for containment of a possible future pandemic are needed. Humanized monoclonal antibodies (mAbs) that neutralize H5N1 virus could be used as prophylaxis and treatment to aid in the containment of such a pandemic.

Methods: Neutralizing mAbs against H5 hemagglutinin were humanized and introduced into C57BL/6 mice (1, 5, or 10 mg/kg bodyweight) one day prior to-, one day post- and three days post-lethal challenge with H5N1 A/Vietnam/1203/04 virus. Efficacy was determined by observation of weight loss as well as survival.

Results: Two mAbs neutralizing for antigenically variant H5N1 viruses, A/Vietnam/1203/04 and A/Hong Kong/213/03 were identified and humanized without loss of specificity. Both antibodies exhibited prophylactic efficacy in mice, however, VN04-2-huG1 performed better requiring only 1 mg/kg bodyweight for complete protection. When used to treat infection VN04-2-huG1 was also completely protective, even when introduced three days post infection, although higher dose of antibody was required.

Conclusion: Prophylaxis and treatment using neutralizing humanized mAbs is efficacious against lethal challenge with A/Vietnam/1203/04, providing proof of principle for the use of passive antibody therapy as a containment option in the event of pandemic influenza.

Figure 1

Humanization of mouse mAbs VN04-2 and VN04-3. A, Diagrammatic representation of the expression vector used to create chimeric antibodies; CL and CH refer to the constant regions of the human Kappa light and human IgG1, respectively; L refers to the leader sequence. B, ELISA to show presence of human constant regions, antibodies bound to immunosorbent plates were detected using secondary antibodies specific for human IgG and mouse IgG. Following addition of TMB substrate absorbance was measured at 450 nm.

Figure 2

Prophylactic efficacy of VN04-2- and VN04-3-huG1 in mice. Mice were challenged with a lethal dose (10 MLD₅₀) of fully virulent A/Vietnam/1203/04 24 h after the introduction of 1, 5, or 10 mg/kg bodyweight of antibody. The percentage of initial body weight after challenge is indicated for VN04-2-huG1 (A) and VN04-3-huG1 (B) periodically over 15 days. Each data point represents the average of 5 mice. Survival of challenged mice was observed for 21 days after challenge and indicates the level of protection from mortality (C).

Figure 3

Therapeutic efficacy of VN04-2-huG1 in mice. Mice were inoculated with a lethal dose (10 MLD₅₀) of A/Vietnam/1203/04 virus 24 h, followed by the introduction of 1, 5, or 10 mg/kg bodyweight of VN04-2-huG1 antibody one (A and B) and three (C and D) days post infection. The percentage of initial body weight was monitored periodically over 15 days (B and D) and each data point represents the average of 5 mice. Survival of mice was observed for 21 days following infection and indicates the level of protection from mortality (A and C).