IFN-I responses in I-Aα^-/-mice.

<p>A) I-Aα^-/-and B6 mice were infected with aSFV and serum samples were collected 24 h post infection and tested for IFN-Iα levels. B) Splenocytes from naïve I-Aα^-/- and B6 mice were tested for cell surface expression of IFN-I receptor. C) Splenocytes from B6, I-Aα^-/- and IFN-αR1 ^-/- mice were incubated <i>in vitro</i> with 1000 U/ml of r-IFN-β (black line) or medium alone (filled grey) for 12 hours and tested for cell surface expression of CD69 and CD25. The expression profile of B220+ cells is shown.</p

I-Aα^-/- cell line supports increased viral replication compared to the B6 cell line generated in parallel.

<p>10⁶ cells from B6 (open symbols) and I-Aα^-/- (solid symbols) fibroblast lines were infected with aSFV (MOI of 10 or 0.1 pfu/cell) and culture supernatants were assayed 24 h later for virus titres by plaque assay.</p

Defective MHC-II expression and Ab response in I-Aα^-/- mice.

<p>A) Cell surface expression of MHC-II on CD19⁺ cells from B6 and I-Aα^-/- mice. B) B6 and I-Aα^-/- mice were immunised with gamma-irradiated SFV and sera analysed for Ab responses and isotype switching.</p

Enhanced virus replication in tissues of I-Aα^-/- mice.

<p>Knockout and wild-type mice were infected i.v with 10³ pfu/mouse of ECT-Moscow (A) or WNV (Sarafend) (B) and animals were monitored for clinical symptoms. Tissues were harvested at day 3 post infection for ECT-Moscow infected animals and day 8 post infection for WNV infected animals and virus titres were estimated using plaque assays.</p

Viral replication in vitro.

<p>Splenocytes and peritoneal macrophages obtained from B6 (open symbols) and I-Aα^-/- (solid symbols) mice support high levels of alphaviruses replication in vitro. A) Splenocytes and macrophages were infected in vitro with aSFV using MOI of 0.5 pfu/cells. B) Macrophages were infected with RRV, Sindbis, and Bebaru using MOI of 0.5 pfu/cells. Culture supernatants were sampled 24 and 48 h post infection and virus titres were estimated using plaque assay.</p

Susceptibility of I-Aα^-/- mice to aSFV infection.

<p>Mice were infected i.v with aSFV and animals were monitored for clinical symptoms and mortality for a period of 21 days (no changes were observed after day 8). A) Both I-Aα^-/- and B6 mice were compared for their susceptibility to aSFV (10³ or 10⁷ pfu/mouse) infection. B) Various MHC-II knockout mice (I-Aα^-/-, I-Aβ^-/- and I-Aαxβ^-/-) and F1 of (I-Aα^-/- x I-Aβ^-/-) were compared for their susceptibility to aSFV (10⁷ pfu/mouse) infection.</p

Tissue virus titres.

<p>B6 (open symbols) and I-Aα^-/- (solid symbols) mice were infected with aSFV (10³ pfu/mouse). Tissues were harvested at days 1 and 2 post infection and virus titres were estimated using plaque assay.</p

Somatic cells of I-Aα^-/- mice support high levels of virus replication.

<p>Females B6 and I-Aα^-/- mice were lethally irradiated and reconstituted with bone marrow cells from female donors of either B6 or I-Aα^-/- mice. Bone marrow chimeras were infected i.v with aSFV (10³ pfu/mouse) and tissue virus titers were estimated at day 1 post-infection using plaque assay.</p

CoVaccine HT™ adjuvant elicits significantly higher anti-HA antibody titres than Freund’s adjuvant.

<p>Sheep (n = 5) were immunised SC with rHA (200 µg) in complete FA or CoVaccine HT™ (CV). Sheep were boosted similarly every two weeks (five boosts indicated by arrows) with rHA in incomplete FA or CV. Pre-immune (time 0) or hyperimmune serum samples were analysed for anti-rHA IgG via ELISA (1/50, 000 dilution) (A) and HAI (B). Data are represented as the mean ± SEM. Two-way repeated-measures ANOVA with Bonferroni post-test was applied to evaluate significance which is denoted as thus: * = P<0.05, ** = P<0.01, *** = P<0.001, ns = not significant.</p

Ovine anti-HA serum elicited with CoVaccine HT™ adjuvant show greater <i>in vivo</i> potency as compared to that by Freund’s adjuvant.

<p>Mice (n = 6) were challenged with 500 TCID₅₀ PR8 and twenty-four hours later therapeutically administered dose equivalents of 1000 (A), 500 (B), 250 (C) or 50 µl (D) of pooled hyperimmune serum (1 ml, IP) from sheep that received rHA antigen in CV (i) or FA (ii). Control groups of mice (n = 6) received non-immune serum (Ei) or PBS (Eii) twenty-four hours following viral challenge. Mice reaching a predetermined endpoint of 20% weight loss (dotted line) were euthanased as indicated by arrows. In each panel, data show percentage weight loss of individual mice. Survival curves of mice are also shown (iii). Mantel-Cox survival analysis was performed on survival curves; significance between survival curves is denoted as thus: * = P<0.05, ** = P<0.01, *** = P<0.001, ns = not significant.</p