Universal stabilization of the influenza hemagglutinin by structure-based redesign of the pH switch regions

Abstract

For an efficacious vaccine immunogen, influenza hemagglutinin (HA) needs to maintain a stable quaternary structure, which is contrary to the inherently dynamic and metastable nature of class I fusion proteins. In this study, we stabilized HA with three substitutions within its pH-sensitive regions where the refolding starts. An X-ray structure reveals how these substitutions stabilize the intersubunit β-sheet in the base and form an interprotomeric aliphatic layer across the stem while the native prefusion HA fold is retained. The identification of the stabilizing substitutions increases our understanding of how the pH sensitivity is structurally accomplished in HA and possibly other pH-sensitive class I fusion proteins. Our stabilization approach in combination with the occasional back mutation of rare amino acids to consensus results in well-expressing stable trimeric HAs. This repair and stabilization approach, which proves broadly applicable to all tested influenza A HAs of group 1 and 2, will improve the developability of influenza vaccines based on different types of platforms and formats and can potentially improve efficacy.

Protein Nanoparticle-Mediated Delivery of Recombinant Influenza Hemagglutinin Enhances Immunogenicity and Breadth of the Antibody Response

“…Our model HA antigen, H1 from A/California/7/2009 (Sino Biological), was His-tagged at the C-terminus, expressed in HEK293T cells, and lacked the transmembrane domain and cytoplasmic tail of native hemagglutinin. The protein also lacks an engineered trimerization domain, and size exclusion chromatography (SEC) comparing the elution profiles of this soluble H1 shows elution profiles consistent with previously reported monomers of H1 72 , 73 and other HA subtypes (H3, H5), 72 − 75 rather than their trimers ( Figure SI-4 ). Although HA trimers may present quaternary epitopes not found in monomers and thereby elicit antibodies from a broader repertoire of B cell clones, 76 , 77 we aim to construct H1–E2 using HA monomers to facilitate synthesis, which is an important consideration for future nanoparticle vaccine scale-up.…”

Antigen spacing on protein nanoparticles influences antibody responses to vaccination

“…Our strategy was similar to those used to stabilize HA stem and coronavirus Spike S2 antigens by deleting undesired domains and then repairing the resulting conformational changes or instabilities with stabilizing mutations or topological rearrangements (Yassine et al 2015;Corbett et al 2019;Impagliazzo et al 2015;Hsieh et al 2021;Bowen et al 2022). We focused on restoring the native-like closed trimeric arrangement of the displayed RBDs by strengthening the interactions between protomers, an approach that has been used to stabilize other oligomeric antigens in native-like conformations (Impagliazzo et al 2015;Ellis et al 2022;Milder et al 2022;Joyce et al 2016;Che et al 2023;Stewart-Jones et al 2021;Hsieh et al 2022;Chuang et al 2017;de Taeye et al 2015;Rutten et al 2018). Trimer closure required both computationally designed hydrophobic interactions at the trimer interface and a rigid, disulfide-mediated connection to an underlying trimerization domain.…”

Antigenic drift expands viral escape pathways from imprinted host humoral immunity

“…HA ectodomain (or head-only) sequences (see below for accession numbers) were cloned into a pVRC8400 backbone containing a C-terminal 3C cleavage site followed by a His tag and AviTag. The full-length ectodomain recombinant HA trimers included three mutations, H26W/K51I/E103I, shown to increase expression and stability of pre-fusion HA and express full-length trimers without the requirement of a trimerization tag 46 . Antibodies and HAs were produced by production in 293F cells or Expi-293F cells, respectively, using PEI (3:1 ratio) for transfection of 293F cells and ExpiFectamine (ThermoFisher, #A14525) for transfection of Expi-293F cells according to the manufacturer’s suggestion.…”