Expression of Acr/Ag85B fusion protein in Vrep-Acr/Ag85B confirmed by Western blot.

<p>BHK cells were electroporated with 15μg of plasmid replicons or were not electroporated (mock), lysates were prepared 18 hours later, and a Western blot was performed using anti-FLAG antibody as described in Materials and Methods.</p

Multifunctional CD4+ T cells persist for at least 10 weeks post immunization with Vreps.

<p>Mice were immunized twice at weeks 0 and 3 with Vrep-Luc or Vrep-Acr/Ag85B vaccines as described in Materials and Methods and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136635#pone.0136635.t001" target="_blank">Table 1</a>. A) Representative dot plots showing lymphocytes gated as CD3+ CD4+ T cells secreting IFN-γ, TNF-α or IL-2 in response to recall antigen. After gating, Boolean analyses allow determination of concurrent expression of these cytokines. Splenocytes were harvested at either 3 or 10 weeks post-immunization and Acr-specific (B) and Ag85B-specific (C) CD4+ T cells producing IFN-γ, TNF-α and/or IL-2 were assessed by ICS after subtraction of values for media controls in each case. Data shown are numbers of cytokine secreting CD4+ T cells per million CD4+ T cells in splenocytes from individual mice (n = 5) as mean ± SEM in a single experiment that is representative of three such experiments. * p < 0.05, + p < 0.01, ++ p < 0.001 by Student’s t-test. Pie charts under the bar graphs depict relative percentages of CD4+ T cells in each vaccine group that produced all three of these cytokines (3+), two of these cytokines (2+) or a single cytokine (1+), after exposure to recall antigen and were created using the SPICEv4.1.5 and PESTLEv1.5.2 software programs (a gift from M. Roederer, Vaccine Research Center, NIAID/National Institutes of Health).</p

Immunization Protocols.

<p>DNA vaccines were administered IM as described in Materials and Methods, such that each animal received a total of 60μg of designated vaccine at week 0 and again at week 3. Vrep vaccines were given IM as described in Materials and Methods, such that each animal received a total of 20μg of designated vaccine at week 0 and again at week 3 or week 6. See figure legends for specific details within each experiment.</p><p>Immunization Protocols.</p

CD4+ and CD8+ T cell IFN-γ responses induced by Vrep vaccination persist at least up to 10 weeks post immunization.

<p>Mice were immunized twice at weeks 0 and 3 with Vrep-Luc or Vrep-Acr/Ag85B vaccines as described in Materials and Methods and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136635#pone.0136635.t001" target="_blank">Table 1</a>. Splenocytes were harvested at 3 or 10 weeks post immunization and Acr and Ag85B-specific CD4+ (A) and CD8+ (B) T cell responses were assessed by IFN-γ ELISpot. Data shown are mean counts of spot forming cells (SFCs) ± SEM from pooled splenocyte samples (n = 5) in a single experiment and are representative of three such experiments. Student’s t-test was used for all group comparisons.</p

Vrep-Acr/Ag85B is immunogenic for CD4+ and CD8+ T cell responses.

<p>Mice were immunized twice at weeks 0 and 3 with Vrep or conventional DNA vaccines as described in Materials and Methods and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136635#pone.0136635.t001" target="_blank">Table 1</a>. The different immunization groups are denoted by the initial vaccine given at week 0 followed by the second vaccine given at week 3 (eg. DNA-control:DNA-Acr/Ag85B). Splenocytes were harvested at 3 wk post-immunization and both Acr and Ag85B-specific CD4+ (A) and CD8+ (B) T cell responses were assessed by IFN-γ ELISpot assay. Data shown are mean counts of spot forming cells (SFCs) ± SEM from pooled splenocyte samples (n = 5) in a single experiment and are representative of three such experiments, * p < 0.05, + p < 0.01, by Students t-test.</p

Increasing the time interval between homologous Vrep-DNA immunizations does not increase the magnitude of T cell IFN-γ responses in spleens and lungs.

<p>Mice were immunized twice at 3 or 6-week intervals with Vrep vaccines and splenocytes and lung lymphocytes were harvested at 10 weeks after immunization for assay of splenic and pulmonary Acr and Ag85B-specific CD4+ T (A) and CD8+ T (B) cell responses by IFN-γ ELISpot, as described in Materials and Methods and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136635#pone.0136635.t001" target="_blank">Table 1</a>. Data shown are SFCs/million lymphocytes from pooled splenocytes or lung samples (n = 5) in one experiment that is representative of three such experiments and are shown as mean SFCs ± SEM. * p ≤ 0.05. + p ≤ 0.01. ++ p = 0.001 by Student’s t-test.</p

Transfer of convalescent serum or B cells from 17D-204 immunized mice to naïve mice partially protects against challenge with wtYFV.

<p>5–6 week old mice were immunized s.c. with 17D-204. (A) serum was harvested and screened by PRNT₈₀ for 17D-204 and Ang71 neutralizing antibody at the indicated days post immunization [***, p = 0.0001 paired t-test]. (B-D) On d21 following immunization, spleens, lymph nodes and sera were collected. CD19+ cells were enriched according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005786#ppat.1005786.s002" target="_blank">S2 Fig</a> and adoptively transferred into naive 8 week old mice. Some mice received passive transfer of sera and total splenocytes from 17D-204 or mock vaccinated animals. Twenty-four hours after the transfer, mice were challenged with Ang71 and monitored daily for (B) cumulative weight loss (C) maximum weight loss and (D) survival. Weight is expressed as a percentage of weight on d0 of challenge [ns, not significant; **, p≤0.005; ***, p≤0.0005; One-way ANOVA with Tukey's multiple comparisons test]. Refer to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005786#ppat.1005786.t001" target="_blank">Table 1</a> for statistics comparing percent of survival and numbers of animals. These data are the combined results of two independent experiments.</p

Immunization with 17D-204 protects mice against cytokine storm associated with virulent wtYFV infection.

<p>5–6 week old mice received 17D-204 immunization or mock vaccination. Mice in the groups receiving Ang71 were infected on d21 after 17D-204 immunization, at approximately 8–9 weeks of age. On the indicated days p.i., serum was harvested and cytokine levels were determined by multiplex Luminex bead assay. Mock groups were collected at a 4d p.i. only. Samples for the Ang71 group were collected on d2, d4, and d5 p.i. Samples for the 17D-204-Ang71 group were collected on d1, d2, d3, and d4 p.i. Samples were collected from three separate experiments. Displayed data are n = 5 for mock, n = 3 for the other groups.</p

17D-204-specific T cells are polyfunctional.

<p>5–6 week old mice were immunized with 17D-204. On d7 or d27 post immunization DLNs and spleens were harvested and the intracellular cytokine response was measured for (A-B) CD4+ T cells responding to <i>in-vitro</i> restimulation with the YFII-E peptide and (C-D) CD8+ T cells responding to <i>in-vitro</i> restimulation with YFI-E and YFI-NS3 peptides. Total CD4+ T cells were grouped by their ability to respond by producing multiple cytokines; (A-B) IFNγ, TNFα and IL-4 [Single, Double, or Triple]. CD8+,CD107a+ T cells (C-D) were grouped by their ability to produce IFNγ, TNFα and IL-2 [None, Single, Double, or Triple]. The results are representative of two experiments (n = 3 mock, n = 6 17D-204). A and C are representative dot plots. Percentages in A and C are percent of total CD4+ (A) or total CD8+ (C) cells. B and D display the mean percentages across all samples.</p

Complete protection against wtYFV requires serum and CD4+ T cells.

<p>Cell populations were magnetically enriched according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005786#ppat.1005786.s002" target="_blank">S2 Fig</a>, mixed as indicated and adoptively transferred into naive eight week old mice. Some groups also received passive transfer of serum. Control groups received passive transfer of serum and total splenocytes from 17D-204 immunized or mock vaccinated animals. Twenty-four hours after the transfer, mice were challenged with Ang71 and monitored daily for (A -B, D) weight and (C and E) survival. Weight is expressed as a percentage of weight on d0 of challenge [ns, not significant; ***, p≤0.0005; One-way ANOVA with Tukey's multiple comparisons test]. Refer to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005786#ppat.1005786.t001" target="_blank">Table 1</a> for statistics comparing percent of survival and numbers of animals. These data are the combined results of two independent experiments. These results were acquired simultaneously with those depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005786#ppat.1005786.g005" target="_blank">Fig 5</a>, and control groups are shared with those depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005786#ppat.1005786.g005" target="_blank">Fig 5</a>.</p