A Computationally Optimized Broadly Reactive Antigen Subtype-Specific Influenza Vaccine Strategy Elicits Unique Potent Broadly Neutralizing Antibodies against Hemagglutinin

Computationally optimized broadly reactive Ags (COBRA) targeting H1 elicit a broad cross-reactive and cross-neutralizing Ab response against multiple H1N1 viral strains. To assess B cell breadth, Mus musculus (BALB/c) Ab-secreting cells elicited by a candidate COBRA hemagglutinin (HA) (termed P1) were compared with Ab-secreting cells elicited by historical H1N1 vaccine strains. In addition, to evaluate the Ab response elicited by P1 HA at increased resolution, a panel of P1 HA-specific B cell hybridomas was generated following immunization of mice with COBRA P1 and the corresponding purified mAbs were characterized for Ag specificity and neutralization activity. Both head- and stem-directed mAbs were elicited by the P1 HA Ag, with some mAbs endowed with Ab-dependent cell-mediated cytotoxicity activity. P1 HA-elicited mAbs exhibited a wide breadth of HA recognition, ranging from narrowly reactive to broadly reactive mAbs. Interestingly, we identified a P1 HA-elicited mAb (1F8) exhibiting broad hemagglutination inhibition activity against both seasonal and pandemic H1N1 influenza strains. Furthermore, mAb 1F8 recognized an overlapping, but distinct, epitope compared with other narrowly hemagglutination inhibition-positive mAbs elicited by the P1 or wild-type HA Ags. Finally, P1 HA-elicited mAbs were encoded by distinct H chain variable and L chain variable gene segment rearrangements and possessed unique CDR3 sequences. Collectively, the functional characterization of P1 HA-elicited mAbs sheds further insights into the underlying mechanism(s) of expanded Ab breadth elicited by a COBRA HA-based immunogen and advances efforts toward design and implementation of a more broadly protective influenza vaccine.