The H5N1 virus is the most likely candidate for an emerging pandemic strain. Owing to the anticipated rapid spread of the pandemic virus, together with the time required to produce a vaccine that matches the circulating strain, the first wave of the pandemic may have passed in many countries before a significant amount of pandemic vaccine is available. The success of national pandemic preparedness strategies may depend on providing appropriate H5N1 pre-pandemic vaccines (i) in a proactive controlled manner (ii) that induce immunologic memory and demonstrate cross-reactivity in the whole population, including the elderly, and (iii) with a proven safety profile. Evidence for the safety of the MF59 adjuvant is provided not only by the present trial, but also from a review of the database (>14,000 clinical trial participants and >30 million distributed doses post-licensure) for the interpandemic influenza vaccine Fluad®
, which differs from the H5N1 vaccine only in its HA content and viral strain composition (2×7.5 μg or 2×15 μg H5N1 versus 1×45 μg HA seasonal viral strains). It has been well established that repeated yearly vaccination with Fluad®
does not lead to increased reactogenicity or other side effects 
, and the post-marketing MF59 safety database showed no increase in spontaneous reports of adverse events compared with conventional influenza vaccines. While little is published on immunopotentiators for pre-pandemic vaccines, a recent study of an H5N1 vaccine using a novel adjuvant in non-elderly adults 
indicates a comparable immune response to that obtained with MF59. However, the novel adjuvant appeared to result in a higher incidence of mild to moderate injection-site pain 
. In contrast, in the current study both 7.5 μg and 15 μg doses of MF59-adjuvanted H5N1 vaccine were equally well tolerated and induced neutralizing antibody responses in non-elderly and elderly adults. Long-term safety assessments of the vaccine beyond the 6-month follow-up period of this study are in progress.
As was the case in previous H5N1 vaccine trials 
, some of our study participants were not immunologically naïve at baseline. The microneutralization results in the 18–60 year age group are consistent with findings from earlier studies indicating that approximately 1–3% of subjects have H5N1 antibody at baseline 
. The higher incidence of H5N1 antibodies in the elderly may reflect increased natural exposure or seasonal vaccination (in particular, the H1N1 component). Others have shown that approximately 34% of recipients of seasonal influenza vaccine have cross-reactive neutralizing antibody to H5N1 
. Since immune responses in baseline seronegative participants were similar to those in the population as a whole, the inclusion of a small number of baseline seropositive individuals does not affect the interpretation of the results.
Regardless of antigen content, the good immune response observed after two primary injections of adjuvanted H5N1 vaccine exceeded all requirements of the European Regulatory Authority 
. More than 76% of the individuals were seroprotected or achieved an MN titer ≥40. Although correlates of protection for MN titers against H5N1 virus infections remain unknown, neutralizing antibody titers ≥40 are increasingly recognized as protective 
. The proportion of younger adults (<60 years) with neutralizing antibody titers ≥40 after the second 7.5 μg H5N1 vaccination compared favorably with figures reported in studies using up to 90 μg of non-adjuvanted 
or 30 μg of aluminum-adjuvanted H5N1 vaccine in individuals aged 18–40 years 
As expected, seroprotection rates fell significantly 6 months after primary vaccination but increased after the booster dose to levels higher than those achieved 3 weeks after the second vaccination, indicating a memory response in both elderly and non-elderly adults. As the timing of the next pandemic influenza outbreak is unknown, the memory response may be more important than persistence, as subsequent exposure to the pandemic vaccine or wild-type virus could trigger a full immunologic response.
A heterologous immune response against H5N1/turkey/Turkey/1/05 (NIBRG-23) was detectable after the primary and booster vaccinations, indicating that there is cross-reactivity between the clade 1 and clade 2 strains. Increased heterologous immune responsiveness against antigenically drifted strains presents a significant public health advantage in the event of a pandemic outbreak. We have shown elsewhere that, among primed subjects, protective cross-reactive antibody titers to diverse H5N1 virus variants can be achieved within 7 days after administration of a single dose of M59-adjuvanted vaccine [Stephenson, manuscript submitted]. In addition, the availability of a 7.5 μg formulation of MF59-adjuvanted influenza vaccine would allow a threefold increase in the number of subjects who could be vaccinated compared with seasonal trivalent influenza vaccines, enabling production of higher quantities of vaccine, in pre-pandemic and pandemic situations, as encouraged by the World Health Organization 
In conclusion, H5N1/Vietnam/1194/2004 influenza vaccine adjuvanted with MF59 can be safely used as a pre-pandemic vaccine. Primary vaccination of non-elderly and elderly adults induces a sufficient immune response and cross-reactivity against the clade 2 H5N1/turkey/Turkey/05 strain, and booster vaccination leads to a strong and sustained immunologic response. A low-dose antigen formulation (7.5 μg) resulted in a comparable seroprotection benefit when compared with a higher dose (15 μg). These results, in conjunction with the extensive safety data on the MF59 adjuvant, suggest that this vaccine would be a suitable choice for proactive priming in advance of pandemic influenza.