Weight loss and fever responses in Wistar-Furth rats treated with T-705.

<p>Results from the dose-determination study (A, C) and the delayed-treatment study (B, D) are shown. For each graph, the shaded area represents the 14 days of T-705 treatment. The black lines represent the mean +/− SEM of the infected, treated surviving rats (n = 33 in A and C; n = 39 in B and D). The red line represents the mean +/− SEM of the infected, untreated control rats (n = 12 in A and C; n = 6 in B and D). Three rats from each experiment were infected, treated, and succumbed to infection. Their weight loss and temperature data are plotted separately on each panel (blue, orange, and green lines). The x-axis is plotted through day 16 only in order to make the earlier time points more clearly visible.</p

Survival of ZH501 RVFV-infected Wistar-Furth rats after treatment with T-705.

<p>(A) Results from T-705 dose-determination study. Groups of 12 rats were infected with RVFV by aerosol exposure, with an average presented dose of 50 pfu/rat. Within 1 hour after infection, twice-daily (BID) T-705 administration was given at the indicated doses. After 14 days (shaded area), drug treatment was removed, and the rats were monitored for another 28 days, as indicated by the split x-axis. (B) Results from T-705 delayed-treatment study. Groups of 6–12 rats were infected by aerosol exposure, with an average presented dose of 20 pfu/rat. All treated rats received 100 mg/kg of T-705 starting at the indicated times post-infection and continuing for 14 days (shaded area), followed by an additional 28 days of monitoring for survival (split x-axis). For ease of visual identification, the T-705 treatment period is indicated by gray shading from days 0–14 post-infection. For the delayed treatment groups, the treatment periods are shifted by 12, 24, or 48 hours, which is not indicated on the graph. In both experiments, all groups of T-705 treated rats survived significantly longer when compared to the untreated control rats (Log-Rank test p<0.0001).</p

Tissue viral loads during T-705 efficacy studies in Wistar-Furth rats.

<p>Plaque assays were used to measure levels of infectious virus within the indicated tissues. Data from both the dose-determination study and the delayed-treatment study are combined in this figure. The black circles represent the tissue viral load from rats that were infected with RVFV but did not receive T-705 treatment (n = 6–12 per tissue; horizontal line is the mean). For the T-705-treated rats, 92% survived (n = 66), and tissues taken at necropsy from the surviving rats did not have detectable tissue viral loads by plaque assay (data not shown). A total of 6 T-705 treated rats from both studies died. The tissue viral loads from these 6 rats are shown as individual data points (red stars; red horizontal line is the mean).</p

IgG antibody responses during T-705 efficacy studies in Wistar-Furth rats.

<p>Total IgG levels were measured using an indirect antigen-capture ELISA, and the data are expressed as the SumOD of each sample. The cutoff value is indicated by the horizontal dotted line, which represents 3 standard-deviations above the control wells. (A) Dose-determination study, and (B) Delayed-treatment study. For the untreated groups on each panel, serum was taken at necropsy when the rats were moribund (between 4–6 days post-infection). For the surviving rats within the T-705 treated groups (black circles), serum samples were taken at necropsy at the end of the study (day 42). Serum was available for 4 of the 6 T-705 treated rats that died (indicated by the red squares on each graph). Unpaired t-tests were used to compare each T-705 treated group with the untreated control group. Level of significance is indicated by the number of asterisks.</p

Pathological changes in T-705-treated Wistar-Furth rats.

#<p> = average score of the degree of severity of pathological lesions, where 0 = no pathology and 3 = severe pathology; +/− SEM.</p

Vaccinated macaques have reduced viral RNA loads and viral replication in nasal washes and lungs.

<p>Three weeks post final vaccination (week 15) control and vaccinated macaques are challenged with 10^7.4 PFU of A/California/04/2009 virus (n = 8/group). A) Bronchioalveolar lavage (BAL) viral RNA titers in control and vaccinated macaques (p<0.0079, Mann-Whitney U test). <i>In situ</i> hybridization with influenza virus specific, ³⁵S-labeled riboprobes was used to localize challenge virus RNAs in the B) right accessory and C) right caudal lung lobes of control and vaccinated macaques at day 3 post infection (n = 3/group). Viral RNA signals are evident as collections of black silver grains over cells. Animal numbers are noted in the upper portion of each micrograph.</p

LT-MA DNA vaccine induces systemic and mucosal antibodies in NHP against HA, M2e, and NP.

<p>Cynomolgus macaques (n = 8) received 3 immunizations with the LT-MA DNA vaccine at 0, 6, and 12 weeks. Sera was collected from vaccinated macaques at various time-points and analyzed for the presence of IgG by ELISA and hemagglutinin inhibition (HI) assay. A) IgG antibody responses against the vaccine components M2e, NP and the representative HA H1N1-A/New Caledonia/20/99 (NC99) in the serum. Dashed line represents an HI titer of 1:40. B) IgG antibody responses in the bronchioalveolar lavage (BAL) collected at various time-points against the vaccine components M2e, NP and HA (NC99). C) Serum HI titers against NC99 virus. D) IC₅₀ titers of vaccinated macaque serum against the matching vaccine strain NC99 and the heterologous unmatched strains PR8 and CA09. Neutralization assays were performed on serum samples taken from vaccinated macaques two weeks after the final immunization.</p

DNA immunization induces influenza-specific T cell responses.

<p>A) IFN-<b>γ</b> analysis was performed on peripheral blood mononuclear cells. Vaccine-induced responses to specific influenza antigens 2 weeks post third vaccination (week 14). Mean response for each animal is plotted (n = 8/group). Error bars represent SEM. B) Responses of vaccinated animals to influenza peptide pools corresponding to subsets of HA and NP as well as M2e at 2 weeks post third vaccination (week 14). C) Total IFN-<b>γ</b> responses against HA, NP, and M2e in vaccinated and control animals at 2 weeks post third vaccination (week 14). Mean of duplicate samples is plotted; error bars represent SEM. Responses after third immunization are significantly elevated over baseline; p = 0.0008 as calculated by Mann-Whitney U test.</p

Vaccinated macaques displayed more rapid immune response and less inflammation.

<p>A) Inflammatory cytokines were assayed by Bio-Plex using BAL obtained from vaccinated and control macaques on day 3 and day 7 following CA09 challenge (day 3 n = 8 per group, day 7 n = 5 per group). Percentage of B) CD4+ and CD8+ T cells (P = 0.018), C) B cells, and D) macrophages in BAL obtained from vaccinated and control macaques at day 3 post-challenge. *P < 0.05, Mann-Whitney U test. Refer to <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189780#pone.0189780.s003" target="_blank">S2 Fig</a></b> for a gating scheme of representative samples.</p

Protection from influenza correlates with NP-specific T cell responses.

<p>Correlation between viral RNA titer (CEID₅₀ equivalents/ml) on day 3 post-infection and A) NP-specific, B) HA-specific, C) M2e- specific ELISpot IFN-<b>γ</b> T cell responses prior to challenge at 2 weeks post third vaccination (week 14) were determined by Spearman-rank correlation tests.</p