We therefore undertook RCT evaluation of the possible direct effects of prior heterologous TIV receipt on Apdm09 disease risk in ferrets as the ideal alternate model of human influenza infection. Although none of the animals became moribund or so severely ill as to require euthanasia, ferrets immunized with two doses of 2008�C09 TIV did show significantly worse clinical, virologic and pathological features following pandemic H1N1 infection compared to placebo recipients. As originally powered to show, vaccinated animals Salvianolic-acid-B experienced significantly greater weight loss relative to baseline following infection, maximally different from placebo at Ch+5. Nasal wash titers did not differ, but vaccinated animals showed significantly higher lung virus titers. Consistent with lung virus findings, lung inflammation was also increased more than 2.5-fold in vaccinated compared to placebo animals at Ch+5 although with fewer animals sacrificed on that day the difference fell just short of statistical significance. Inflammatory indicators were, however, significantly higher at Ch+5 compared to Ch+14 in the vaccinated animals and showed no change across that period in placebo recipients. Lung cytokines showed a similar pattern. Although illness activity levels appeared similar between groups based on categories of induced playfulness, these may have been of insufficient resolution to reflect clinical differences in lung disease, manifest otherwise through significant loss of appetite and weight. By day 14 post-challenge animals in both groups had recovered. In human studies, a doubling of the risk of medically-attended pandemic H1N1 illness was observed among 2008�C09 TIV recipients, but an increase in the risk of hospitalization was not shown. This was broadly interpreted to suggest increased risk of acquiring infection per se whereas here we report increased disease severity among influenza-na? ��ve, systematically-infected ferrets. As such, our findings in ferrets may not replicate the experience in humans. It is worth noting, however, that the source population in the human observational studies was patients seeking medical care. Although illness severity among outpatient visits was not specifically compared between vaccinated and unvaccinated participants, subjects had experienced influenza-like illness severe enough to prompt medical consultation within 
one week of illness onset, and that outcome was significantly increased among the vaccinated. In that regard, the pattern of acute worsening of Apdm09 illness during the first week of infection in vaccinated ferrets, followed by subsequent recovery by day 14, is consistent with increased outpatient but not hospitalization risk observed in vaccinated humans. As such, this ferret RCT suggests that earlier findings from observational studies in humans cannot be dismissed on the basis of methodological bias alone and that direct mechanistic explanations should be sought. Whether these findings may be product-specific or may also apply to other TIV products has yet to be separately assessed in follow-up studies. To date, hypotheses about biological mechanisms to explain increased Apdm09 risk among prior TIV recipients have included both direct and indirect vaccine effects. Indirect mechanisms include the infection block hypothesis whereby effective seasonal vaccine may prevent the more robust and complex cross-protective immunity against heterologous viruses afforded by seasonal infection, such as through cell-mediated responses to conserved internal virus components. Other epidemiological investigators have favoured this hypothesis, or related Lomitapide Mesylate variations, but in Appendix G of our original publication we demonstrated these indirect hypotheses.