Traditional influenza vaccines primarily induce a narrow antibody response that offers no protection against heterosubtypic infections. Murine studies have shown that T cells can protect against a broad range of influenza strains. However, ferrets are a more potent model for studying immune correlates of protection in influenza infection. We therefore set out to investigate the role of systemic and respiratory T cells in the protection against heterosubtypic influenza A infections in ferrets. H1N1-priming induced systemic and respiratory T cells that responded against pandemic H2N2 and correlated with reduced viral replication and disease. CD8-positive T cell responses in the upper and lower respiratory tract were exceptionally high. We additionally confirmed that H2N2-responsive T cells are present in healthy human blood donors. These findings underline the importance of the T cell response in influenza immunity and show that T cells are a potent target for future universal influenza vaccines.

Until universal influenza vaccines become available, pandemic preparedness should include developing classical vaccines against potential pandemic influenza subtypes. We here show that addition of SWE adjuvant, a squalene-in-water emulsion, to H7N9 split influenza vaccine clearly enhanced functional antibody responses in ferrets. These were cross-reactive against H7N9 strains from different lineages and newly emerged H7N9 variants. Both vaccine formulations protected in almost all cases against severe pneumonia induced by intratracheal infection of ferrets with H7N9 influenza; however, the SWE adjuvant enhanced protection against virus replication and disease. Correlation analysis and curve fitting showed that both VN- and NI-titers were better predictors for protection than HI-titers. Moreover, we show that novel algorithms can assist in better interpretation of large data sets generated in preclinical studies. Cluster analysis showed that the adjuvanted vaccine results in robust immunity and protection, whereas the response to the non-adjuvanted vaccine is heterogeneous, such that the protection balance may be more easily tipped toward severe disease. Finally, cluster analysis indicated that the dose-sparing capacity of the adjuvant is at least a factor six, which greatly increases vaccine availability in a pandemic situation.

Key Points • CD41 T-cell responses in 2 patients with acquired TTP.• CUB2 domain-derived core peptides are recognized by CD4 1 T cells present in 2 patients with acquired TTP.Acquired thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder resulting from the development of autoantibodies against ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). HLA-DRB1*11 provides a risk factor for developing acquired TTP. Pulsing of antigen-presenting cells from HLA-DRB1*11-and HLA-DRB1*03-positive individuals with ADAMTS13 resulted in presentation of peptides derived from the CUB2 domain of ADAMTS13 with core sequences FINVAPHAR or ASYILIRD. Here, we assessed whether FINVAPHAR-or ASYILIRD-reactive CD4 IntroductionThe autoimmune disorder thrombotic thrombocytopenic purpura (TTP) is characterized by the development of autoantibodies against ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) in previously healthy individuals.

Acquired thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder that results from the development of auto-antibodies against ADAMTS13. Recent research suggests that HLA-DRB1*11 provides a risk factor for the development of acquired TTP. Previous work in our department identified ADAMTS13 derived peptides that are presented on MHC class II (Sorvillo et al. 2013). Pulsing of dendritic cells from HLA typed healthy donors showed preferential presentation of the CUB-2 domain derived peptides "FINVAPHAR" and "ASYILIRD" on HLA-DRB1*11 and HLA DRB1*03 respectively. In this study we screened peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with acquired TTP for reactive CD4+ T cells against the previously identified peptides. The presence of CD4+ T cells recognizing FINVAPHAR or the ASYILIRD peptide was addressed by flow cytometry. PBMCs were stimulated for 24 hours and up regulation of CD40L was visualized on CD4+ T cells. Stimulation with Staphylococcus aureus enterotoxin B (SEB) and overlapping peptides from cytomegalovirus (CMV) pp65 protein were used as controls. Several samples derived from patients in the acute phase of the disease were analyzed in this study. A high frequency of FINVAPHAR responsive CD4+ T cells was identified by monitoring the up regulation of CD40L on CD4+ T cells in a sample from a HLA-DRB1*1104 patient. Four percent of the CD4+ T cells were activated in response to the FINVAPHAR peptide. A control peptide in which the anchor-residues of the FINVAPHAR peptide were modified did not induce activation of CD4+ T cells. Incubation with full length ADAMTS13 also induced activation of CD4+ T cells although to a lesser extent. No FINVAPHAR reactive CD4+ T cells were observed after stimulation in HLA-DRB1*1104 positive healthy individuals. PBMC samples from HLA-DRB1*0301 positive patients were screened for CD4+ T cells reactive with the ASYILIRD peptide. ASYILIRD positive CD4+ T cells were detected in samples of 2 DRB1*0301 positive patients. In one patient 0.5% reactive CD4+ T cells were detected after stimulation with the ASYILIRD peptide. In samples derived from a second patient 1.5% of CD4+ T cells reactive against the ASYILIRD peptide were detected. Stimulation with a control peptide in which the anchor residues were modified did not result in activation of CD4+ T cells in both patients. Stimulation with SEB and CMV derived peptides resulted in activation of CD4+ T cells in the samples analyzed. No CD40L+ CD4+ T cells were observed after incubation with full length ADAMTS13, this may be due to insufficient processing of ADAMTS13 under our experimental conditions. Taken together our data provide the first evidence for the presence of ADAMTS13 reactive CD4+ T cells in acquired TTP. Based on our results we propose that FINVAPHAR and ASYILIRD CD4+ T cells are involved in the onset and/or relapse of acquired TTP. Disclosures No relevant conflicts of interest to declare.

In 1957 the world was subjected to a pandemic caused by an influenza A virus of the subtype H2N2. Although the virus disappeared in 1968, H2 viruses continue to circulate in avian reservoirs.