Neutralization titers against CHIKV-S27 and CHIKV-IND/NL10.

<p>Neutralization titers against CHIKV-S27 and CHIKV-IND/NL10.</p

Detection of antigen expression.

<p>BHK-21 cells were infected with the respective candidate MVA vaccines and 24 hours later cells were fixed in methanol/acetone (1∶1) and stained for specific expression of CHIKV E1 (upper panel), E2 (middle panel) or MVA antigens (lower panel). The respective antigens were detected using rabbit polyclonal serum specific against E1, E2, and MVA as indicated. The staining confirmed the specific expression of the respective antigens and their purity. *Images were contrast enhanced in Adobe Photoshop.</p

Survival of mice after vaccination and challenge infection with CHIKV.

<p>(<b>A</b>). Mice (n = 8) were immunized intra-muscularly with MVA-6KE1, MVA-E3E2, MVA-E3E26KE1 or MVA-wt. Subsequently, the mice were challenged intra-peritoneally with 1000 TCID₅₀ CHIKV-S27. The survival rates of the mice after challenge are depicted as Kaplan-Meier curves. Differences between the curves were determined by the log-rank test. (<b>B, C, D</b>). Vial RNA copies were determined in spleen (B), liver (C) and brain (D) samples of animals that succumbed to the infection and survivors (day 14 post challenge). (<b>E</b>) Neutralizing antibody titers were determined on day 0, 21, 63, 70, and 77. Animals were immunized on day 0 and 21 and challenged on day 63. A clear booster response is seen after challenge, where a higher response was measured in MVA-6KE1 immunized group. The results are expressed as TCID₅₀ equivalents per gram of tissue; *indicates a statistically significant result (<i>P</i><0.05) and ** indicates highly significant results (<i>P</i><0.001) as determined by the <i>Student's t test</i>.</p

Histopathology of several tissues staining positive for CHIKV antigen.

<p>Panels A–E show representative staining of the liver, spleen, and brain of CHIKV infected AG129 mice. Hematoxylin and eosin-stained sections of the liver from all groups showed no abnormalities (A; 40× objective). Endothelial and Kupffer cells stained positive with anti-CHIKV capsid antibody (B, 40× objective). Massive depletion of lymphocytes was observed in the spleen of animals that died after challenge with CHIKV (C; HE staining, 40× objective). Antigen was mainly located in endothelial cells in the spleen (D). Epithelia cells of the choroid plexus (E) in the brain were scarcely stained and no antigen was found in the neuropil of the brain. Examples of positively stained cells are indicated by block arrows.</p