Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design

Rappuoli, Rino; Bottomley, Matthew J.; D’Oro, Ugo; Finco, Oretta; De Gregorio, Ennio

The identification of human mAbs able to bind conserved epitopes in the HA stem region of several flu strains belonging to different subtypes can be considered the first step toward the discovery of an ideal immunogen for a universal flu vaccine. These Abs are precious tools that can be used both for cocrystal structure studies to precisely identify the neutralizing epitopes in the HA stem region and for binding assays to isolate the most appropriate immunogen to induce a broad protection. Two different groups have recently taken advantage of such anti-HA stem Abs and the cognate structurally derived epitope mapping information to rationally design and screen a series of novel immunogens, leading to selection of a stable immunogen containing the appropriate conformational epitopes from the HA stem (Impagliazzo et al., 2015; Yassine et al., 2015) . A team led by Nabel and Graham have again taken advantage of ferritin nanoparticles to present a refined HA antigen comprised of a stem-only immunogen, which was rationally designed and stabilized in a trimeric "headless" state (Yassine et al., 2015) . In the study, the design strategy was driven by prior human immunology studies and detailed structural knowledge that showed that the immunogenically subdominant stem region of HA contains highly conserved protective conformational epitopes recognized by human mAbs with multisubtype HA recognition profiles broader than those targeting the more variable head region (Throsby et al., 2008; Ekiert et al., 2009 Ekiert et al., , 2011 Sui et al., 2009; Corti et al., 2011a; Dreyfus et al., 2012; Friesen et al., 2014) . New structural studies by x-ray crystallography and cryoelectron microscopy informed an itera-tive design pathway that resulted in seven generations of H1 subtype HA-stabilized stem immunogens (termed H1-SS), with the last designed form presenting the desired trimeric structure and the epitope recognized by the broadly neutralizing human mAbs. When this recombinant antigen was fused to ferritin, it assembled on the nanoparticle surfaces (termed H1-SS-np), which were confirmed to possess the desired antigenic features. More importantly, in preclinical immunization studies, H1-SS-np induced a higher Ab response against the HA stem compared with the classical trivalent inactivated flu vaccine, with cross-reactivity not only to many H1 strains but also to distant subtypes from both group 1 (H2, H5, and H9) and group 2 (H3 and H7). Although neutralization could be observed only for the homologous and two other heterologous H1 strains, vaccination with H1-SS-np resulted in the complete protection of mice and partial protection of ferrets against lethal viral challenge using the H5N1 heterosubtype. The latter finding was further supported by passive transfer of immunoglobulins from the immunized mice, which protected naive mice against H5N1 challenge (Yassine et al., 2015) , suggesting that anti-HA stem Abs can have a protective effect via a different mechanism than virus neutralization.