IM010 IDO1 and TNFRSF1B Knockout (KO) Pilot Part 1: MLD 50 analysis Part 2: Tissue collection for further analysis IDO1 HPAI H5N1 and SARS common bottleneck Primarily DC and macrophage May regulate type I IFN expression May facilitate expansion of Treg cells TNFRSF1B HPAI H5N1 and SARS common eigengene Role in apoptosis

MLD 50 Analysis Inoculation 3 mice per inoculation group 0.1, 1, 10 and 100 PFU dosages Measurements Time frame: 14 days MLD 50 calculation (Reed and Muench method) Survival (mean time to death) Group body weight changes

GenotypeMLD 50 Wild-type0.18 PFU IDO1 KO0.56 PFU TNFRSF1B3.16 PFU Mouse Lethal Dose 50 (MLD 50 ) for VN1203 Wild-type, IDO1 KO and TNFRSF1B KO Ratio of KO to Mock: Note that this is not a hugely impressive difference in MLD50.

MLD 50 Survival and Weight Loss Data: 1 PFU Wild-type, IDO1 KO and TNFRSF1B KO All wild-type died Two IDO1 KO died All TNFRSF1B KO lived Rapid weight loss in wild-type Intermediate weight loss in IDO1 KO No weight loss in TNFRSF1B KO

No differences in weight loss or survival at higher dosages (10 and 100 PFU) Subtle differences at lower dosage (0.1 PFU)

MLD 50 Mean Time to Death per Dosage Wild-type, IDO1 KO and TNFRSF1B KO

Tissue Harvest Groups Inoculation 3 mice per inoculation group, mock and VN PFU dosage Measurements Time frame: 6 days Harvest tissues on day 2 and before death (days 5-6) Body weights Survival Lung titer RNA for microarray (optional) Histopathology (optional)

Tissue Harvest Groups Weight Loss Dosage: 1000 PFU Wild-type, IDO1 KO and TNFRSF1B KO Survival Wild-type: All animals died on day 5; lung tissues harvested only on day 2  IDO1 KO: All animals euthanized on day 6; lung tissues harvested on days 2 and 6 TNFRSF1B KO: One animal died between days 5-6 and 2 were euthanized on day 6; lung tissues harvested on days 2 and 6 Longer survival and significantly reduced body weight loss for both KO genotypes

Tissue Harvest Groups Virus titer Dosage: 1000 PFU Wild-type, IDO1 KO and TNFRSF1B KO No differences in virus titers were observed at the early time point.

For Comparison and Perspective… Wild-type mice infected with VN1203-wild type, VN1203-NS1trunc124 or VN1203-PB1-F2del NS1-trunc PFU PB1-F2del 1000 PFU

For Comparison and Perspective… Wild-type mice infected with VN1203-wild type, VN1203-NS1trunc124 or VN1203-PB1-F2del NS1-trunc PFU PB1-F2del 1000 PFU