Potent adjuvant effects of novel NKT stimulatory glycolipids on hemagglutinin based DNA vaccine for H5N1 influenza virus

• α-Galactosylceramide analog, C34, is a potent adjuvant for consensus HA DNA vaccine.
• C34 induced higher production of HA-specific antibodies than non-adjuvanted group.
• Antisera from C34-treated mice showed broader and greater neutralizing activities.
• C34-adjuvanted group displayed higher survival rate upon lethal challenge of H5N1 virus.

H5N1 influenza virus is a highly pathogenic virus, posing a pandemic threat. Previously, we showed that phenyl analogs of α-galactosylceramide (α-GalCer) displayed greater NKT stimulation than α-GalCer. Here, we examined the adjuvant effects of one of the most potent analogs, C34, on consensus hemagglutinin based DNA vaccine (pCHA5) for H5N1 virus. Upon intramuscular electroporation of mice with pCHA5 with/without various α-GalCer analogs, C34-adjuvanted group developed the highest titer against consensus H5 and more HA-specific IFN-γ secreting CD8 cells (203 ± 13.5) than pCHA5 alone (152.6 ± 13.7, p < 0.05). Upon lethal challenge of NIBRG-14 virus, C34-adjuvanted group (84.6%) displayed higher survival rate than pCHA5 only group (46.1%). In the presence of C34 as adjuvant, the antisera displayed broader and greater neutralizing activities against virions pseudotyped with HA of clade 1, and 2.2 than pCHA5 only group. Moreover, to simulate an emergency response to a sudden H5N1 outbreak, we injected mice intramuscularly with single dose of a new consensus H5 (pCHA5-II) based on 1192 full-length H5 sequences, with C34 as adjuvant. The latter not only enhanced the humoral immune response and protection against virus challenge, but also broadened the spectrum of neutralization against pseudotyped HA viruses. Our vaccine strategy can be easily implemented for any H5N1 virus outbreak by single IM injection of a consensus H5 DNA vaccine based on updated HA sequences using C34 as an adjuvant.