Glycan‐masking hemagglutinin antigens from stable CHO cell clones for H5N1 avian influenza vaccine development

Refocusing of B‐cell responses can be achieved by preserving the overall fold of the antigen structure but selectively mutating the undesired antigenic sites with additional N ‐linked glycosylation motifs for glycan masking the vaccine antigen. We previously reported that glycan‐masking recombinant H5 hemagglutinin (rH5HA) antigens on residues 83, 127, and 138 (g127 + g138 or g83 + g127 + 138 rH5HA) elicited broader neutralizing antibodies and protection against heterologous clades/subclades of high pathogenic avian influenza H5N1 viruses. In this study, we engineered the stably expressing Chinese hamster ovary (CHO) cell clones for producing the glycan‐masking g127 + g138 and g83 + g127 + g138 rH5HA antigens. All of these glycan‐masking rH5HA antigens produced in stable CHO cell clones were found to be mostly oligomeric structures. Only the immunization with the glycan‐masking g127 + g138 but not g83 + g127 + g138 rH5HA antigens elicited more potent neutralizing antibody titers against four out of five heterologous clades/subclades of H5N1 viral strains. The increased neutralizing antibody titers against these heterologous viral strains were correlated with the increased amounts of stem‐binding antibodies, only the glycan‐masking g127 + g138 rH5HA antigens can translate into more protection against live viral challenges. The stable CHO cell line‐produced glycan‐masking g127 + g138 rH5HA can be used for H5N1 subunit vaccine development.