Design of Nanoparticulate Group 2 Influenza Virus Hemagglutinin Stem Antigens That Activate Unmutated Ancestor B Cell Receptors of Broadly Neutralizing Antibody Lineages

Abstract

Influenza vaccines targeting the highly conserved stem of the hemagglutinin (HA) surface glycoprotein have the potential to protect against pandemic and drifted seasonal influenza viruses not covered by current vaccines. While HA stem-based immunogens derived from group 1 influenza A viruses have been shown to induce intragroup heterosubtypic protection, HA stem-specific antibody lineages originating from group 2 may be more likely to possess broad cross-group reactivity. We report the structure-guided development of mammalian-cell-expressed candidate vaccine immunogens based on influenza A virus group 2 H3 and H7 HA stem trimers displayed on self-assembling ferritin nanoparticles using an iterative, multipronged approach involving helix stabilization, loop optimization, disulfide bond addition, and side-chain repacking. These immunogens were thermostable, formed uniform and symmetric nanoparticles, were recognized by cross-group-reactive broadly neutralizing antibodies (bNAbs) with nanomolar affinity, and elicited protective, homosubtypic antibodies in mice. Importantly, several immunogens were able to activate B cells expressing inferred unmutated common ancestor (UCA) versions of cross-group-reactive human bNAbs from two multidonor classes, suggesting they could initiate elicitation of these bNAbs in humans. IMPORTANCE Current influenza vaccines are primarily strain specific, requiring annual updates, and offer minimal protection against drifted seasonal or pandemic strains. The highly conserved stem region of hemagglutinin (HA) of group 2 influenza A virus subtypes is a promising target for vaccine elicitation of broad cross-group protection against divergent strains. We used structure-guided protein engineering employing multiple protein stabilization methods simultaneously to develop group 2 HA stem-based candidate influenza A virus immunogens displayed as trimers on self-assembling nanoparticles. Characterization of antigenicity, thermostability, and particle formation confirmed structural integrity. Group 2 HA stem antigen designs were identified that, when displayed on ferritin nanoparticles, activated B cells expressing inferred unmutated common ancestor (UCA) versions of human antibody lineages associated with cross-group-reactive, broadly neutralizing antibodies (bNAbs). Immunization of mice led to protection against a lethal homosubtypic influenza virus challenge. These candidate vaccines are now being manufactured for clinical evaluation.

Cross‐reactive antibody‐dependent cellular cytotoxicity antibodies are increased by recent infection in a household study of influenza transmission

“…However, we observed no significant difference between infected and uninfected participants for the G2 stem antibodies by ADCC responses at baseline, fold‐change after infection or ELISA assays despite clear and specific detection of G2 stem antibody responses . The generation of G2 stem antibodies is notoriously more difficult because of an additional N‐glycosylation at HA1‐Asn38 restricting access, resulting in the isolation of far fewer G2 stem monoclonal antibodies, and additional modifications required to stabilise the G2 stem headless structure for vaccination models …”

“…22 The generation of G2 stem antibodies is notoriously more difficult because of an additional N-glycosylation at HA1-Asn38 restricting access, 23,24 resulting in the isolation of far fewer G2 stem monoclonal antibodies, 25 and additional modifications required to stabilise the G2 stem headless structure for vaccination models. 26 Antibody-dependent cellular cytotoxicity responses are reportedly highly cross-reactive, and avian influenza-specific ADCC antibodies can be found in healthy unexposed individuals 7,27 and enriched for the HA stem. 8,28 In our study, ADCC activity towards conserved NP, cross-reactive H7-HA, seasonal HA and NA proteins was boosted by recent H1N1 infection.…”

Vaccine innovations for emerging infectious diseases—a symposium report

“…As examples, immunization with an engineered, stable HA stem protected against group 1 influenza A viruses in both mouse and nonhuman primate models, and HA‐stem nanoparticles elicited heterosubtypic protection in rodent models . More recently, group 2 HA stem–based influenza A antigens displayed on self‐assembling nanoparticles showed homosubtypic protection in mice and elicited bnAbs associated with broad cross‐neutralizing activity …”

“…63 More recently, group 2 HA stem-based influenza A antigens displayed on selfassembling nanoparticles showed homosubtypic protection in mice and elicited bnAbs associated with broad cross-neutralizing activity. 64 Wilson also discussed efforts to develop a malaria vaccine, focusing on vaccines that target the sporozoite stage. Many bnAbs target the P. falciparum CSP.…”

Designed Nanoparticles Elicit Cross-Reactive Antibody Responses To Conserved Influenza Virus Hemagglutinin Stem Epitopes

“…The epitopes of some of these antibodies, including human monoclonal antibodies FI6V3 and CR6261, have also been structurally mapped (17,28,30,36,37,(46)(47)(48). This has facilitated interest in structure-guided efforts to engineer trimeric HA2 stem immunogens that elicit stem antibodies (29,35,(49)(50)(51)(52).…”