Divergent Requirement of Fc-Fcγ Receptor Interactions for In Vivo Protection against Influenza Viruses by Two Pan-H5 Hemagglutinin Antibodies

Abstract

Recent studies have shown that Fc-Fc␥ receptor (Fc␥R) interactions are required for in vivo protection against influenza viruses by broadly reactive antihemagglutinin (HA) stem, but not virus strain-specific, anti-receptor binding site (RBS), antibodies (Abs). Since only a few Abs recognizing epitopes in the head region but outside the RBS have been tested against single-challenge virus strains, it remains unknown whether Fc-Fc␥R interactions are required for in vivo protection by Abs recognizing epitopes outside the RBS and whether the requirement is virus strain specific or epitope specific. In the present study, we therefore investigated the requirements for in vivo protection using two pan-H5 Abs, 65C6 and 100F4. We generated chimeric Abs, 65C6/IgG2a and 100F4/IgG2a, which preferentially engage activating Fc␥Rs, and isogenic forms, 65C6/D265A and 100F4/D265A, which do not bind Fc␥R. Virus neutralizing activity, binding, antibody-dependent cellular cytotoxicity (ADCC), and in vivo protection of these Abs were compared using three H5 strains, A/Shenzhen/406H/2006 (SZ06), A/chicken/Shanxi/2/2006 (SX06), and A/chicken/Netherlands/14015526/2014 (NE14). We found that all four chimeric Abs bound and neutralized the SZ06 and NE14 strains but poorly inhibited the SX06 strain. 65C6/IgG2a and 100F4/IgG2a, but not 65C6/D265A and 100F4/D265A, mediated ADCC against target cells expressing HA derived from all three virus strains. Interestingly, both 65C6/IgG2a and 65C6/D265A demonstrated comparable protection against all three virus strains in vivo; however, 100F4/IgG2a, but not 100F4/D265A, showed in vivo protection. Thus, we conclude that Fc-Fc␥R interactions are required for in vivo protection by 100F4, but not by 65C6, and therefore, protection is not virus strain specific but epitope specific.IMPORTANCE Abs play an important role in immune protection against influenza virus infection. Fc-Fc␥R interactions are required for in vivo protection by broadly neutralizing antistem, but not by virus strain-specific, anti-receptor binding site (RBS), Abs. Whether such interactions are necessary for protection by Abs that recognize epitopes outside RBS is not fully understood. In the present study, we investigated in vivo protection mechanisms against three H5 strains by two pan-H5 Abs, 65C6 and 100F4. We show that although these two Abs have similar neutralizing, binding, and ADCC activities against all three H5 strains in vitro, they have divergent requirements for Fc-Fc␥R interactions to protect against the three H5 strains in vivo. The FcFc␥R interactions are required for in vivo protection by 100F4, but not by 65C6. Thus, we conclude that Fc-Fc␥R interactions for in vivo protection by pan-H5 Abs is not strain specific, but epitope specific.KEYWORDS influenza viruses, in vivo protection, monoclonal antibodies

The Vestigial Esterase Domain of Haemagglutinin of H5N1 Avian Influenza A Virus: Antigenicity and Contribution to Viral Pathogenesis

“…Of these, 100F4 was shown to have both prophylactic and therapeutic efficacies which were able to outperform a five-day treatment regime with 10 mg/kg of oseltamivir in mice challenged with H5N6 or H5N8 [ 13 ]. To assess the importance of ADCC function for 100F4 protection against influenza, Wang and colleagues generated chimaeric versions of 100F4, either with a mouse IgG2a Fc region which preferentially stimulates ADCC or with a D265A mutation which is unable to stimulate ADCC [ 31 ]. When tested in vivo, 100F4/IgG2a, but not 100F4/D265A, was able to confer protection in mice infected with the H5 strains A/Shenzhen/406H/2006, A/chicken/Netherlands/14015526/2014, or A/chicken/Shanxi/2/2006, suggesting that the ADCC pathway was essential for 100F4 protection.…”

“…Although the VE-targeting mAbs 100F4 and 46B8 are specific for different viral types of influenza, many similarities exist between them. When tested in vitro, 100F4 was able to directly neutralise A/Shenzhen/406H/2006 and A/chicken/Netherlands/14015526/2014 [ 31 ] by binding to the virus and preventing low-pH-triggered membrane fusion after endocytosis [ 12 ]. 46B8 was also able to neutralise IBV via the same mechanism.…”

The Vestigial Esterase Domain of Haemagglutinin of H5N1 Avian Influenza A Virus: Antigenicity and Contribution to Viral Pathogenesis

“…When tested in vitro, 100F4 was able to directly neutralise A/Shenzhen/406H/2006 and A/chicken/Netherlands/14015526/2014 [29] by binding to IAV and preventing low-pH triggered membrane fusion after entry into the cell with IAV [12]. 46B8 was also able to neutralise IBV via the same mechanism.…”

“…Of these, 100F4 was shown to have both prophylactic and therapeutic efficacies which were able to outperform a 5 day treatment regime with 10mg/kg of oseltamivir in mice challenged with H5N6 or H5N8 [13]. To assess the importance of ADCC function for 100F4 protection against influenza, Wang and colleagues generated chimaeric versions of 100F4 either with a mouse IgG2a Fc region which preferentially stimulates ADCC or with a D265A mutation which is unable to stimulate ADCC [29]. protection.…”

Contribution of Fc-dependent cell-mediated activity of a vestigial esterase-targeting antibody against H5N6 virus infection

“…Collectively, the information currently available suggests that VE-binding antibodies generally inhibit virus entry by blocking membrane fusion and are able to neutralize different virus clades within the same virus subtype [39]. All three reported mAbs 100F4 [40], 46B8 [37] and H3v-47 [34] were also capable of initiating ADCC, although ADCC was only shown to be essential for 100F4 but was not yet assessed in 46B8 and H3v-47.…”