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..

Vaccine development for circulating influenza viruses, particularly for the H3N2 subtype, remains challenging due to consistent antigenic drift. Computationally optimized broadly reactive antigen (COBRA) technology has proven effective for broadening influenza hemagglutinin (HA)-elicited antibody responses compared to wild-type immunogens.

Neuraminidase (NA) is an important surface protein on influenza virions, playing an essential role in the viral life cycle and being a key target of the immune system. Despite the importance of NA-based immunity, current vaccines are focused on the hemagglutinin (HA) protein as the target for protective antibodies, and the amount of NA is not standardized in virion-based vaccines. Antibodies targeting NA are predominantly protective, reducing infection severity and viral shedding. Recently, NA-specific monoclonal antibodies have been characterized, and their target epitopes have been identified. This review summarizes the characteristics of NA, NA-specific antibodies, the mechanism of NA inhibition, and the recent efforts towards developing NA-based and NA-incorporating influenza vaccines.

Computationally optimized broadly reactive antigens (COBRAs) are a next-generation universal influenza vaccine candidate. However, how these COBRAs induce antibody responses when combined with different adjuvants has not previously been well-characterized. Therefore, we performed in vivo studies with an HA-based H1 COBRA, Y2, and an NA-based N1 COBRA, N1-I, to assess this effect for the H1N1 subtype. We tested the adjuvants AddaVax, AddaS03, CpG, and Alhydrogel. AddaS03 performed the best, eliciting high IgG titers and hemagglutination inhibition (HAI) activity for Y2 immunizations. Interestingly, serum antibody epitopes were relatively similar across adjuvant groups. Moreover, following N1-I immunization with these adjuvants, AddaS03 also elicited the highest IgG and neuraminidase inhibition (NAI) titers against the 2009 pandemic virus, A/California/07/2009 (A/CA/09). These results inform adjuvant selection efforts for H1 and N1 COBRA HA and NA antigens in a mouse model.

Among circulating influenza viruses in humans, H3N2 viruses typically evolve faster than other subtypes and have caused severe illness and deaths in millions of people since emerging in 1968. Computationally optimized broadly reactive antigen (COBRA) technology is one strategy to broaden vaccine-elicited antibody responses among influenza subtypes. In this study, we determined the structural integrity of an H3N2 COBRA HA, TJ5, and, as nearly all humans have pre-existing immunity to H3N2 influenza viruses, we probed the antigenic profile of several H3N2 COBRA HAs by assessing recognition of these immunogens by human B cells and monoclonal antibodies (mAbs). Of three recently described COBRA H3 HA antigens (TJ5, NG2, and J4), we determined that TJ5 and J4 HA proteins recognize pre-existing B cells (from the 2017-2018 vaccine season) more effectively than NG2 HA and a wild type Hong Kong/4801/2014 protein. H3 HA-specific human mAbs recognize wild type and COBRA HA proteins, and have functional activity against a broad panel of H3N2 viruses. mAb TJ5-5 recognizes TJ5 and J4 HA proteins, but has poor recognition of NG2 HA, similar to the global B cell analysis. To probe these recognition differences and to verify the structural integrity of the TJ5 HA protein, we determined a 3.4 Å structure via cryo-electron microscopy of TJ5-5 complexed with the TJ5 HA, which revealed residues important to the differential binding. Overall, these studies determined that COBRA H3 HA proteins have correct antigenic and structural features, and are recognized by B cells and mAbs isolated from seasonally vaccinated humans.