Antigenic characterisation of human monoclonal antibodies for therapeutic use against H7N9 avian influenza virus

Abstract

Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1500 human deaths and millions of poultry culling. Despite large-scale poultry vaccination, H7N9 AIVs continue to circulate among poultry in China and pose a threat to human health. Previously, we isolated and generated four monoclonal antibodies (mAbs) derived from humans naturally infected with H7N9 AIV. Here, we investigated the haemagglutinin (HA) epitopes of H7N9 AIV targeted by these mAbs (L3A-44, K9B-122, L4A-14 and L4B-18) using immune escape studies. Our results revealed four key antigenic epitopes at HA amino acid positions 125, 133, 149, and 217. The mutant H7N9 viruses representing escape mutations containing Alanine to Threonine at residue 125 (A125T), Glycine to Glutamic acid at residue 133 (G133E), Asparagine to Aspartic acid at residue 149 (N149D), or Leucine to Glutamine at residue 217 (L217Q) showed reduced or completely abolished cross-reactivity with the mAbs, as measured by hemagglutination inhibition (HI) assay. We further assessed the potential risk of these mutants to humans should they emerge following mAb treatment by measuring the impact of these HA mutations on virus fitness and evasion of host adaptive immunity. Here we showed that the L4A-14 mAb had broad neutralizing capability, and its escape mutant N149D had reduced viral stability and human receptor binding and could be neutralized by both post-infection and antigen-induced sera. Therefore, L4A-14 mAb could be a therapeutic candidate for H7N9 AIV infection in humans and warrants further investigation for therapeutic application.IMPORTANCEAvian Influenza virus (AIV) H7N9 continues to circulate and evolve in birds, posing a credible threat to humans. Antiviral drugs have been proven useful for the treatment of severe influenza infections in humans, however, concerns have been raised as antiviral resistant mutants have emerged. Monoclonal antibodies (mAbs) have been studied for both prophylactic and therapeutic applications in infectious disease control and have demonstrated great potential. For example, mAb treatment has significantly reduced the risk of people developing severe disease with SARS-COV 2 infection. In addition to the protection efficiency, we should also consider the potential risk of the escape mutants generated by mAb treatment to public health by assessing their viral fitness and potential to compromise host adaptive immunity. Considering these parameters, we assessed four human mAbs derived from humans naturally infected with H7N9 AIV and showed that the mAb L4A-14 displayed potential as a therapeutic candidate.

Risk Assessment of the newly emerged H7N9 avian influenza viruses

“…The recombinant viruses were generated by reverse genetics with HA and NA from these H7N9 AIVs and six internal segments from PR8 virus (A/Puerto Rico/8/34 [H1N1]). In agreement with our previous reports (9, 20), the H7N9 AH/13 showed comparable binding to both 3SLN and 6SLN receptor analogues (Figure 1) . In comparison to the AH/13, the GD/16 from the epidemic wave five showed significantly stronger binding to 3SLN and a slight increase in binding to 6SLN receptor analogues respectively (∼293-fold increase to 3SLN and ∼2-fold increase to 6SLN).…”

“…We previously isolated and generated four monoclonal antibodies (mAbs) from humans naturally infected with H7N9 A/Anhui/1/2013 (20, 23). The mAb L3A-44, K9B-122, L4A-14 and L4B-18 target HA residues 125 (Alanine (A)), 133 (Glycine (G)), 149 (Asparagine (N)), and 217 (Leucine (L)), respectively.…”

“…To investigate the cause for the reduced cross-reactivity between mAbs and the newly emerged H7N9 AIVs, the HA amino acid sequences of these H7N9 AIVs were analysed. All the newly emerged H7N9 AIVs harbour Threonine (T) at amino acid residue 125 and 151 (Table 2), which introduced an N-linked glycosylation at amino acid residues 123 and 149 (9); such introduction has been shown to abolish the binding to L3A-44 and L4A-14 respectively (20). The drop in HI titre of L4B-18 with the 12 newly emerged H7N9 AIVs might be due to the L217Q substitution in HA, which has been shown to reduce its cross-reactivity with L4B-18.…”