Antigenic Fingerprinting of Antibody Response in Humans following Exposure to Highly Pathogenic H7N7 Avian Influenza Virus: Evidence for Anti-PA-X Antibodies

Abstract

Infections with H7 highly pathogenic avian influenza (HPAI) viruses remain a major public health concern. Adaptation of lowpathogenic H7N7 to highly pathogenic H7N7 in Europe in 2015 raised further alarm for a potential pandemic. An in-depth understanding of antibody responses to HPAI H7 virus following infection in humans could provide important insight into virus gene expression as well as define key protective and serodiagnostic targets. Here we used whole-genome gene fragment phage display libraries (GFPDLs) expressing peptides of 15 to 350 amino acids across the complete genome of the HPAI H7N7 A/Netherlands/33/03 virus. The hemagglutinin (HA) antibody epitope repertoires of 15 H7N7-exposed humans identified clear differences between individuals with no hemagglutination inhibition (HI) titers (<1:10) and those with HI titers of >1:40. Several potentially protective H7N7 epitopes close to the HA receptor binding domain (RBD) and neuraminidase (NA) catalytic site were identified. Surface plasmon resonance (SPR) analysis identified a strong correlation between HA1 (but not HA2) binding antibodies and H7N7 HI titers. A proportion of HA1 binding in plasma was contributed by IgA antibodies. Antibodies against the N7 neuraminidase were less frequent but targeted sites close to the sialic acid binding site. Importantly, we identified strong antibody reactivity against PA-X, a putative virulence factor, in most H7N7-exposed individuals, providing the first evidence for in vivo expression of PA-X and its recognition by the immune system during human influenza A virus infection. This knowledge can help inform the development and selection of the most effective countermeasures for prophylactic as well as therapeutic treatments of HPAI H7N7 avian influenza virus. IMPORTANCEAn outbreak of pathogenic H7N7 virus occurred in poultry farms in The Netherlands in 2003. Severe outcome included conjunctivitis, influenza-like illness, and one lethal infection. In this study, we investigated convalescent-phase sera from H7N7-exposed individuals by using a whole-genome phage display library (H7N7-GFPDL) to explore the complete repertoire of post-H7N7-exposure antibodies. PA-X is a recently identified influenza virus virulence protein generated by ribosomal frameshifting in segment 3 of influenza virus coding for PA. However, PA-X expression during influenza virus infection in humans is unknown. We identified strong antibody reactivity against PA-X in most H7N7-exposed individuals (but not in unexposed adults), providing the first evidence for in vivo expression of PA-X and its recognition by the immune system during human infection with pathogenic H7N7 avian influenza virus.A vian influenza viruses (AIVs) are mostly restricted to aquatic birds. On occasion, they acquire the capacity to directly infect the respiratory tract of poultry and mammals. Such cross-species transmission often results in a large number of sick birds (chickens and turkeys), as was reported for H7N1 viruses in Italy in 1999, H7N3 viruses in Canada in 2...

A Potent Germline-like Human Monoclonal Antibody Targets a pH-Sensitive Epitope on H7N9 Influenza Hemagglutinin

“…Hemagglutination inhibition assays were conducted with H7N9 HA as antigen and horse red blood cells as indicators using routine methods, as described previously ( Hobson et al., 1972 , Khurana et al., 2016 ). Polyclonal sera from rabbits that were immunized with the trimeric H7N7 or H7N9 HA1 domains (K23, K64, K65, K66) were used as positive controls.…”

“…Viral cytopathic effect (CPE)-based neutralization assay was conducted to determine the neutralizing antibody titers of mAb against live H7N3 and H7N9 viruses as previously described ( Khurana et al., 2016 ). Polyclonal sera from rabbits immunized with H7N7 or H7N9 HA1 (K23, K64, K65, K66), or from sheep immunized with H7N9 HA1 were used as positive controls.…”

AIV polyantigen epitope expressed by recombinant baculovirus induces a systemic immune response in chicken and mouse models

“…The ELISA results indicate that the antibody levels induced by BmNPV-CMV/THB-P10/CTLT increased roughly with the immunization in a dose-dependent manner in both chickens and mice. Although previous studies have shown that the epitope regions selected in this study are critical for the production of neutralizing antibodies [22,24,35,40], a neutralizing assay for AIV should be conducted to test whether these B cell epitopes could induce neutralized antibody in the further.…”

Biosensor-based epitope mapping of antibodies targeting the hemagglutinin and neuraminidase of influenza A virus

“…As previously described, generation of escape mutants has been useful for determining epitopes of the HA/NA, but this approach is time-consuming and involves growing virus which may require special lab facilities and permissions for highly pathogenic strains of IAV and potential gain-of-function mutations (Davis et al, 2014; Schultz-Cherry et al, 2014). Phage display has been used to analyze the antibody repertoires of sera from individuals exposed to A(H5N1) or A(H7N7) IAVs but with low mapping resolution (Khurana et al, 2009; Khurana et al, 2016). X-ray crystallography has been used as another strategy to obtain high-resolution epitope maps of the HA and NA.…”