Protection and mean survival time after passive transfer.

(1)<p>results of two independent experiments.</p

Immunization scheme, VV PRNT₅₀ values prior to YF vaccination and survival of mice.

(1)<p>Geometric mean titer;</p>(2)<p>results of three independent experiments;</p>(3)<p>results of two independent experiments.</p

Plasmid transfer vectors (A, C) and genome structures of MVA-YF (B) and dVV-YF (D).

<p>The plasmid vector pd3-lacZ-mH5-YFprMEco (A) targets the deletion III insertion site in the MVA genome. To obtain recombinant virus (B) without any auxiliary sequences, the transient lacZ/gpt screening marker in the plasmid is flanked by a 220 bp self repeat (R) of one of the MVA flanks that mediates removal of the marker cassette by homologous recombination. The insertion site for the plasmid vector pDW-mH5-YFprMEco (C) is the region between the ORFs D3R and D5R in the wild-type Lister/Elstree virus. The lacZ/gpt marker cassette is located between tandem DNA repeats (R, hatched boxes) to achieve eventual removal of the marker cassette. The resulting recombinant defective virus (D) lacks the uracil DNA glycosylase gene (D4R), and still contains one tandem repeat <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024505#pone.0024505-Staib1" target="_blank">[45]</a>. Both plasmids contain the human codon-optimized YFV prM and E coding region under the control of the early/late vaccinia virus mH5 promoter.</p

Protection studies in Balb/c mice.

<p>Animals were vaccinated i.m. in a single dose scheme with the indicated doses of (A) MVA-YF, (B) dVV-YF or with (C) the positive control YFV-17D (17D) and the negative controls wild-type MVA, defective vaccinia virus (dVV) or buffer (PBS). Mice were challenged i.c. 21 days later with 1×10⁵ TCID₅₀ of YFV-17D vaccine strain and monitored for 14 days. Results are the average of 3 individual experiments.</p

Protection and pre-challenge YFV plaque reduction neutralization titers in mice.

(1)<p>Geometric mean titer;</p>(2)<p>Results of two independent experiments;</p>(3)<p>results of three independent experiments (except dVV);</p>(4)<p>not determined;</p>(5)<p>protection studies 10⁶ TCID₅₀, PRNT studies 10⁷ TCID₅₀;</p>(6)<p>determined at d19.</p

Safety of recombinant candidate vaccines in BALB/c mice.

<p>(A) Animals were injected i.c. with 1×10⁵ to 1×10⁷ TCID₅₀ (only 1×10⁷ TCID₅₀ dose shown) of MVA-YF (bright grey line), dVV-YF (grey line) and the corresponding controls wild-type MVA (dotted line) and dVV (black line) and monitored for 21 days. (B) Mice were injected i.c. with YFV-17D vaccine at doses of 1×10¹ (bright grey line), 1×10² (grey line) or 1×10³ (dotted line), and monitored for 21 days.</p

YFV prME protein expression under permissive (A and B) and non-permissive (C and D) conditions.

<p>(A) Western blot of lysates from chicken cells (DF-1) infected with MVA-YF or the corresponding controls. MVA-YF (Lane 1), wild-type MVA (Lane 2), non-infected DF-1 cells (Lane 3), positive control YFV-17D infected DF-1 cells (17D, Lane 4), YFV-17D prepared from infected HeLa cells (17D control, Lane 5). (B) Western blot of lysates from cVero22 cells infected with dVV-YF or the corresponding controls. dVV-YF (Lane 1), wild-type dVV (Lane 2), non-infected cVero22 cells (Lane 3), positive control YFV-17D infected cVero22 (17D, Lane 4), 17D control (Lane 5). Western blot of mouse muscle cells (Sol8, C) or human cells (HeLa, D) infected with the recombinants or the corresponding controls. MVA-YF (Lane 1), dVV-YF (Lane 2), wild-type MVA (Lane 3), wild-type dVV (Lane 4), non-infected Sol8 (C) HeLa (D) cells (Lane 5), cells infected with YFV-17D (17D, Lane 6), 17D control (Lane 7). The band around 55 kDa marked “E” indicates the YFV envelope protein.</p

Cellular immune response elicited against YFV E-antigen.

<p>(A) FACS analysis of the number of IFN-γ secreting CD4⁺ T-cells after two immunizations with MVA-YF, dVV-YF or the corresponding YFV-17D (17D) positive or wild-type MVA and dVV negative controls. Splenocytes from mice were stimulated with 15mer peptides of the YFV E-protein, E57–71 (E4; black bars), E129–143 (E5; grey bars) and E133–147 (E6; white bars). (B) FACS analysis of the number of IFN-γ secreting CD8⁺ T cells after the two immunizations as indicated above. Splenocytes from mice were stimulated with 9-mer peptides of the YFV E-protein, E60–68 (E1; black bars), E330–338 (E2; grey bars), E332–340 (E3 white bars); ** p<0.001. (C) FACS analysis of cytotoxic killing of peptide-pulsed target cells by specific CD8⁺ T cells. Target cells were loaded with 9mer peptides of the YFV E-protein, E60–68 (E1; black bars), E330–338 (E2; grey bars), E332–340 (E3 white bars). The data are mean values (+/− SD) of two independent experiments.</p