Introduction
In a prior Phase 1 trial we showed that an intranasal (IN) recombinant hemagglutinin (HA) H5 vaccine (rH5; A/Indo/05) nanoemulsion-adjuvanted (rH5 NE) and given on days 1 and 29 effectively primed the immune system. Participants received rH5 NE (25—100 μg; IN; n?=?24), unadjuvanted rH5 (100 μg; IN; n?=?7), or placebo (n?=?6). Only rH5 NE recipients exhibited significant increases in HAI titers after a heterologous intramuscular (IM) H5N1 booster on day 197, confirming successful mucosal priming. Anti-H5 antibody-dependent cell cytotoxicity (ADCC) activity also demonstrated this priming effect. Here, we evaluated whether rH5 NE elicited cross reactive ADCC and antibody-dependent cell phagocytosis (ADCP) responses to diverse H5 HAs and examined contributions of HA head and stalk domains.

Methods
Plasma from days 1, 57, and 225 was analyzed. ADCC was assessed using beads coated with whole, head only, or stalk only H5 HAs incubated with plasma and exposed to a CD16a expressing reporter cell line. ADCP was quantified by THP 1 uptake of antigen-coated AF488—beads. ADCC seroconversion was defined as a?≥?4 fold increase; ADCP seroconversion as a?≥?2 fold increase.

Results
By day 57, 33% (8/24) rH5 NE recipients showed ADCC seroconversion to A/Indo/05 whole HA, rising to 50% after the day-197 IM boost. Unadjuvanted rH5 and placebos seroconverted only after boosting (4/7=57% & 4/6=67%, respectively). Similar trends were seen for A/Viet/04 and A/cow Tx/24. Head specific ADCC seroconversion in rH5 NE reached 46% (11/24) at day 57 and 88% (21/24) at day 225, whereas unadjuvanted rH5 responded only at day 225 (4/7=57%). Placebos showed no responses. Stalk specific ADCC was largely preexisting across all groups, with minimal vaccine induced increases. ADCP trends mirrored ADCC findings.

Conclusion
Intranasal rH5 NE induces cross reactive ADCC and ADCP responses driven primarily by antibodies targeting the H5-head, supporting its capacity to induce effective mucosal priming.