The Pre-Existing Human Antibody Repertoire to Computationally Optimized Influenza H1 Hemagglutinin Vaccines

Abstract

The computationally optimized broadly reactive antigen (COBRA) approach has previously been used to generate hemagglutinin (HA) immunogens for several influenza subtypes that expand vaccine-elicited antibody breadth. As nearly all individuals have pre-existing immunity to influenza viruses, influenza-specific memory B cells will likely be recalled upon COBRA HA vaccination.We determined the epitope specificity and repertoire characteristics of pre-existing human B cells to H1 COBRA HA antigens. Cross-reactivity between wild type HA and H1 COBRA HA proteins were observed at both the oligoclonal B cell level and for a subset of isolated monoclonal antibodies (mAbs). The mAbs bound five distinct epitopes on the pandemic A/California/04/2009 head and stem domains, and the majority of the mAbs had HAI and neutralizing activity against pandemic H1 strains. Two head-directed mAbs, CA09-26 and CA09-45, had HAI and neutralizing activity against a pre-pandemic H1 strain. One mAb, P1-05, targets the stem region of H1 HA proteins, but does not compete with known stem-targeting H1 mAbs. We determined that mAb P1-05 recognizes a recently discovered membrane proximal epitope on HA, the anchor epitope, and we identified similar mAbs using B cell repertoire sequencing. In addition, the trimerization domain distance from HA was critical to recognition of this epitope by P1-05. Overall, these data indicate that seasonally vaccinated individuals possess a population of functional H1 COBRA HAreactive B cells that target head, central stalk, and anchor epitopes, and demonstrate the importance of structure-based assessment of subunit protein vaccine candidates to ensure accessibility of optimal protein epitopes..

Adjuvant-Mediated Differences in Antibody Responses to Computationally Optimized Hemagglutinin and Neuraminidase Vaccines

“…To assess the specific epitopes targeted by serum from adjuvanted Y2 immunizations, we performed competition ELISAs against the wild-type CA09 HA ( Figure 4 and Figure S1 ). We assessed the competition of pooled terminal serum against a panel of previously characterized human monoclonal antibodies (mAbs) that bind both the head and stem domains, including the recently characterized anchor epitope [ 14 ]. Serum competition was observed as a decrease in human mAb-binding signal relative to the mAb only control ( Figure S1 ).…”

“…Human monoclonal antibodies (mAbs) used in competition ELISAs were isolated and characterized previously [ 14 ]. 384-well plates (Greiner Bio-One, Monroe, NC, USA) were coated with CA09 HA diluted to 2 µg/mL in PBS at 4 °C overnight.…”

“…Within the more conserved stem domain, the central stem epitope is a target of group 1-specific, broadly reactive antibodies including CR6261 [ 12 ]. Antibodies that bind the membrane-proximal region of the HA at the anchor epitope have been identified as well [ 13 , 14 ]. Whereas the HA component of each virus varies substantially across strains, the NA component remains relatively stable, accruing mutations only occasionally [ 15 ].…”

Structural insights into the broad protection against H1 influenza viruses by a computationally optimized hemagglutinin vaccine

“…1 and 4a ). We then compared this mutant P1 (P1 N127D) with the unmodified construct (P1 wt) by measuring binding with a set of head-targeting monoclonal antibodies (mAbs) elicited by CA/04/09 11 , 19 (Fig. 4b , Supplementary Fig.…”

“…While these have demonstrated the effectiveness of COBRA HA in both naïve and pre-immune models 10,47 , more data is needed to ascertain how it translates to human populations with more diverse genetic backgrounds and complex exposure histories. Recent studies have provided encouraging hints on this front, however, identifying broadly reactive antibodies in human vaccine cohorts that are cross-reactive with COBRA HAs 19,48 . This suggests that COBRA HAs retain structural elements capable of recalling existing protective antibody responses, while potentially broadening the immune response against additional strains.…”