Four groups of 6 ferrets were immunized intramuscularly with two doses of 15, 7.5, 3.8 or 1.7 µg HA of inactivated split A/H5N1/Vietnam/1194/04 NIBRG-14 (recombinant clade 1 H5N1 engineered by reverse genetics) vaccine adjuvanted with a proprietary oil-in-water emulsion based Adjuvant System 
. The two control groups included ferrets administered with either the Adjuvant System alone or the non-adjuvanted A/Vietnam vaccine (containing 15 µg HA). Ferrets were vaccinated on days 0 and 21 and challenged intratracheally on day 49 with a lethal dose of A/Indonesia/5/2005 virus, 105
(50% tissue culture infective dose). Following the challenge with A/Indonesia all control animals receiving adjuvant alone or non-adjuvanted A/Vietnam vaccine died or were moribund and were euthanized on days 3 or 4 (). In contrast, all ferrets that received two doses of ≥3.8 µg of the adjuvanted A/Vietnam vaccine survived the lethal heterologous challenge. Furthermore, all except one animal survived the challenge in the group of ferrets who received the lowest dose (1.7 µg HA) of the adjuvanted vaccine. Thus overall 96% of animals immunized with adjuvanted H5N1 split vaccine were protected against the lethal challenge with A/Indonesia and survived to the end of the challenge phase on day 5 (see ).
Efficacy of adjuvanted split H5N1-vaccine against a heterologous H5N1 challenge in ferrets.
Moribund animals showed general depression, anorexia, lethargy and exhibited clinical signs of respiratory disease, including dyspnea. All animals that died prematurely showed signs of atypical pneumonia in one or more lung lobes by macroscopic and/or microscopic analysis (data not shown).
High levels of virus replication (≥105.5 TCID50/g tissue) were observed in the lungs of all ferrets immunized with either the Adjuvant System alone or with non-adjuvanted A/Vietnam vaccine. Conversely, in 67% ferrets immunized with the adjuvanted A/Vietnam vaccines, lung virus loads were <102 TCID50/g of lung tissue (). These low virus loads were observed in 80% and 83% of ferrets immunized with the adjuvanted 7.5 µg and 15 µg formulations, respectively. However, there was no overall antigen-dose dependent effect on viral load observed among ferrets immunized with adjuvanted A/Vietnam vaccines (). In general high levels of virus replication in the lung correlated with mortality. Of the animals that died all had a lung virus load ≥8×104 TCID50/g tissue, and there was only one animal with a high viral load (1×105 TCID50/g tissue) who survived until the end of the experiment (day 5 post-challenge).
Both the amount of virus shed into the upper respiratory tract and numbers of animals shedding virus were reduced in vaccinees relative to control animals. A majority (92%) of ferrets inoculated with the Adjuvant System alone or the non-adjuvanted A/Vietnam vaccine shed high levels of virus (>102
/ml) in the upper respiratory tract (throat or nasal swabs) throughout the course of infection. Conversely, only 26% ferrets receiving adjuvanted A/Vietnam vaccines shed virus in throat or nasal swabs and none of the ferrets immunized with the 3.8 or 7.5 µg doses of adjuvanted A/Vietnam vaccines exhibited viral shedding >102
/ml (). Since the probability of viral transmission would likely decrease with reduced virus shedding in the upper respiratory tract 
, our data suggest a potential for vaccination to confer a lower risk of viral transmission, a key property in controlling pandemic virus spread within populations.
Before vaccination, ferrets used in this study were influenza naïve as measured by an ELISA assay for the presence of antibodies specific for nucleoprotein 
. Post-vaccination serological assessments showed that the adjuvanted A/Vietnam vaccine formulations induced neutralizing antibody responses against the homologous A/Vietnam strain with 74% of responders (ferrets with neutralizing antibody titres > 28) compared to 100% non responders in control groups (). Furthermore, the adjuvanted A/Vietnam vaccine induced inter-clade cross-neutralizing antibody responses to the heterologous A/Indonesia clade 2 strain () with 61% responders, while no neutralizing antibody response (<28) was observed in ferrets immunized with the non-adjuvanted A/Vietnam vaccine or the Adjuvant System alone. No antigen-dose dependent effect on neutralizing antibody titres was observed amongst ferrets immunized with adjuvanted A/Vietnam vaccines. Interestingly, all animals without a detectable neutralizing antibody response to A/Vietnam or to A/Indonesia exhibited lung virus load > 8×103
/g tissue, whereas 94% (16/17) of ferrets with anti-A/Vietnam neutralizing antibody response and 93% (13/14) with anti-A/Indonesia responses showed virus loads in their lungs below 102
/g tissue. Of 22 animals surviving until the termination of the experiment at day five post-challenge, 17 (77%) were positive for the induction of an H5N1 neutralizing antibody response. Four of the five non-responder animals that were protected from mortality exhibited reduced virus loads in the lung relative to unvaccinated control animals (ranging from 9×103
/g tissue), suggesting a possible role for vaccine induced cellular immune responses in the control of virus replication.
Neutralizing antibody responses to the vaccine strain (A/Vietnam) and the challenge strain (A/Indonesia) 42 days after first vaccination, i.e 21 days after second vaccination
These results highlight the potential of this adjuvanted split H5N1 candidate vaccine to induce, even with a low dose of antigen (3.8 µg), a strong cross-protective response in ferrets against a lethal challenge with heterologous H5N1 virus from another genetic sublineage and suggest that cross-protection may be mediated at least in part by antigen-induced humoral immunity. However, we cannot rule out a role for cell mediated immune responses in cross-protection in the ferret model. There is evidence that cell-mediated immune responses can be linked to protection against influenza in humans 
and it has been shown in other disease systems that adjuvants can be effective at inducing protective cell mediated immune responses 
. As immunological reagents needed to study T cell responses in ferrets are largely lacking, investigation of influenza vaccine-induced antigen-specific T cell responses will be undertaken in mouse and macaque models and in clinical studies.
Data reported in the literature and other preliminary investigations (unpublished observations) suggest that multiple mechanisms of action can account for the immunostimulatory properties of the adjuvant used in this study. Indeed, in addition to their vehicle properties, oil-in-water based emulsions have been shown to induce local inflammation and to attract immunocompetent cells to the injection site 
. In this context, studies are currently ongoing to elucidate the adjuvanted vaccine's ability to induce cell mediated immune responses.
Other published studies have similarly documented cross-clade protection in ferrets vaccinated with different pandemic vaccine candidates 
. Here we document the first study in which protection against heterologous challenge in ferrets is generated by a candidate pandemic vaccine proven to be safe and immunogenic in humans 
, inducing neutralizing antibodies specific for both the vaccine strain and cross-reactive to the heterologous H5N1 virus from a distant clade. These parallel sets of encouraging data with the same product suggest the development of a safe and effective pre-pandemic vaccine is a realistic goal.