Monoclonal antibodies against the fusion peptide of hemagglutinin protect mice from lethal influenza A virus H5N1 infection.

The HA2 glycopolypeptide (gp) is highly conserved in all influenza A virus strains, and it is known to play a major role in the fusion of the virus with the endosomal membrane in host cells during the course of viral infection. Vaccines and therapeutics targeting this HA2 gp could induce efficient broad-spectrum immunity against influenza A virus infections. So far, there have been no studies on the possible therapeutic effects of monoclonal antibodies (MAbs), specifically against the fusion peptide of hemagglutinin (HA), upon lethal infections with highly pathogenic avian influenza (HPAI) H5N1 virus. We have identified MAb 1C9, which binds to GLFGAIAGF, a part of the fusion peptide of the HA2 gp. We evaluated the efficacy of MAb 1C9 as a therapy for influenza A virus infections. This MAb, which inhibited cell fusion in vitro when administered passively, protected 100% of mice from challenge with five 50% mouse lethal doses of HPAI H5N1 influenza A viruses from two different clades. Furthermore, it caused earlier clearance of the virus from the lung. The influenza virus load was assessed in lung samples from mice challenged after pretreatment with MAb 1C9 (24 h prior to challenge) and from mice receiving early treatment (24 h after challenge). The study shows that MAb 1C9, which is specific to the antigenically conserved fusion peptide of HA2, can contribute to the cross-clade protection of mice infected with H5N1 virus and mediate more effective recovery from infection.

Complementary monoclonal antibody-based dot ELISA for universal detection of H5 avian influenza virus.

BACKGROUND: Rapid diagnosis and surveillance for H5 subtype viruses are critical for the control of H5N1 infection. RESULTS: In this study, H5 Dot ELISA, a rapid test for the detection of avian H5N1 influenza virus, was developed with two complementary H5 monoclonal antibodies. HA sequencing of escape mutants followed by epitope mapping revealed that the two Mabs target the epitope component (189th amino acid) on the HA protein but are specific for different amino acids (189Lys or 189Arg). Gene alignment indicated that these two amino acids are the most frequent types on this position among all of the H5 AIV reported in GeneBank. These two H5 Mabs were used together in a dot ELISA to detect H5 viral antigen. The detection limit of the developed test for multiple clades of H5N1 viruses, including clades 0, 1, 2.1, 2.2, 2.3, 4, 7, and 8, was less than 0.5 hemagglutinin units. The specificity of the optimized dot ELISA was examined by using 100 H5 strains, including H5N1 HPAI strains from multiple clades, 36 non-H5N1 viruses, and 4 influenza B viruses. No cross-reactivity was observed for any of the non-H5N1 viruses tested. Among 200 random poultry samples, the test gave 100% positive results for all of the twelve RT-PCR-positive samples. CONCLUSIONS: Considering that the test is convenient for field use, this H5 Dot ELISA can be used for on-site detection of H5N1 infection in clinical or environmental specimens and facilitate the investigation of H5N1 influenza outbreaks and surveillance in poultry.

Prophylactic and therapeutic efficacy of a chimeric monoclonal antibody specific for H5 haemagglutinin against lethal H5N1 influenza.

BACKGROUND: Recent outbreaks of highly pathogenic H5N1 viruses in humans indicate that no endogenous protection exists in the general population. Vaccination programmes against this new pathogen require synthesis of endogenous antibodies and cannot provide any immediate protection in the event of a pandemic. Passive immunization with humanized neutralizing monoclonal antibodies can prove to be promising in preventing a catastrophic pandemic. METHODS: A murine monoclonal antibody (mAb) 3B1 of immunoglobulin M isotype was switched to a chimeric immunoglobulin G1. BALB/c mice were used to study the protective efficacy of the chimeric mAbs against a lethal H5N1 virus challenge with strains from clades 1 and 2.1. Kinetics of the viral load were determined during the course of the treatment. RESULTS: The chimeric mAb, in passive administration, was able to protect 100% of the mice when challenged with H5N1 strains from clades 1 or 2.1. Prophylaxis at 1 day prior to challenge and treatment at 1 day after challenge with this mAb resulted in the clearance of the virus from the lungs of the infected mice within 6 days post-viral challenge. CONCLUSIONS: Passive immunotherapy using chimeric mAb 3B1 can be an effective tool in both the prophylaxis and treatment of highly pathogenic H5N1 infection, providing the immediate immunity needed to contain a future influenza pandemic.

Combination therapy using chimeric monoclonal antibodies protects mice from lethal H5N1 infection and prevents formation of escape mutants.

BACKGROUND: Given that there is a possibility of a human H5N1 pandemic and the fact that the recent H5N1 viruses are resistant to the anti-viral drugs, newer strategies for effective therapy are warranted. Previous studies show that single mAbs in immune prophylaxis can be protective against H5N1 infection. But a single mAb may not be effective in neutralization of a broad range of different strains of H5N1 and control of potential neutralization escape mutants. METHODS/PRINCIPAL FINDINGS: We selected two mAbs which recognized different epitopes on the hemagglutinin molecule. These two mAbs could each neutralize in vitro escape mutants to the other and in combination could effectively neutralize viruses from clades 0, 1, 2.1, 2.2, 2.3, 4, 7 and 8 of influenza A H5N1 viruses. This combination of chimeric mAbs when administered passively, pre or post challenge with 10 MLD50 (50% mouse lethal dose) HPAI H5N1 influenza A viruses could protect 100% of the mice from two different clades of viruses (clades 1 and 2.1). We also tested the efficacy of a single dose of the combination of mAbs versus two doses. Two doses of the combination therapy not only affected early clearance of the virus from the lung but could completely prevent lung pathology of the H5N1 infected mice. No escape variants were detected after therapy. CONCLUSIONS/SIGNIFICANCE: Our studies provide proof of concept that the synergistic action of two or more mAbs in combination is required for preventing the generation of escape mutants and also to enhance the therapeutic efficacy of passive therapy against H5N1 infection. Combination therapy may allow for a lower dose of antibody to be administered for passive therapy of influenza infection and hence can be made available at reduced economic costs during an outbreak.