Neutralizing Antibody Responses of HA-Vaccinated Mice and Ferrets.

<p>*UD = Undetectable; NA = Not Assessed.</p><p>Neutralization was determined by lentiviral inhibition assay, hemagglutinin inhibition assay, and microneutralization assay. Sera from the indicated mouse and ferret immunizations with the indicated viral antigens by DNA alone or DNA/rAd5 before the viral challenge were evaluated by pseudotyped lentiviral inhibition, hemagglutinin inhibition (HAI), and microneutralization assays (MN). UD represents serum samples with undetectable neutralization activities even at the lowest dilutions, while NA represents samples that were not available, and therefore not assessed. In both mice and ferrets, only HA-containing groups stimulated strong humoral responses.</p

DNA immunization with HA, HA+NP, and HA+NP+M2 induces similar protection after A/Vietnam/1203/2004 virus challenge in mice.

<p>(A) Survival data is shown as a percentage comparing the final animal number at day 21 with the initial animal number in each group. All HA-containing groups showed significant survival compared to controls. There is no statistical difference between the HA, HA+NP and HA+NP+M2 groups (<i>p</i> = 0.317 between HA and HA+NP; <i>p</i> = 0.146 between HA and HA+NP+M2; <i>p</i> = 0.515 between HA+NP and HA+NP+M2 by log-rank test); NP and the control groups were not statistically different from each other. (B) Body weights of the mice were also monitored and the total body weight of all of the surviving animals in each group was compared with the respective initial body weights. As expected, the HA group showed the least amount of body weight loss, with the other HA-containing groups showing similar patterns. However, the NP-immunized group demonstrated severe weight loss, similar to controls.</p

Protection of DNA/rAd5 vaccines encoding HA or HA+NP+M2, but not NP, NP+M2, or M2, against A/Vietnam/1203/2004 virus challenge.

<p>(A) Ferrets immunized three times with DNA followed by a single rAd5 boost were challenged under anesthesia with 10⁷ EID₅₀/ferret of influenza virus A/Vietnam/1203/2004. The animals were monitored 7 days for survival, shown as a percentage comparing the initial animal number to the final animal number in the same group (left panel). There was no statistical difference between the control group and groups immunized with NP, NP+M2, or M2. Both the HA and HA+NP+M2 groups showed 100% survival (right panel), whereas the vector control group showed 0% survival after the viral challenge. There was no statistically significant difference between the HA and HA+NP+M2 groups (<i>p</i> = 1.00), but there was a significant difference between these groups and the control (<i>p</i> = 0.008), by a log-rank test. (B) Body weights of the ferrets were also monitored and the total body weight of all of the surviving animals in each group was compared with the respective initial body weight (left panel). Ferrets immunized with HA and HA+NP+M2 groups showed no weight loss, while the control group ferrets showed rapid weight loss (right panel). The survival and initial animal numbers in each group on the last day of body weight data collection are indicated next to the curve labels. The survival percentage for each group was analyzed statistically by a log-rank test.</p

Expression of immunogens using DNA and rAd5 vectors in cell culture.

<p>(A) Western blot analysis confirmed the expression of HA protein of A/Thailand/1/KAN-1/2004 (lane 2), NP protein of A/PR/8/34 (lane 3) and A/Thailand/1/KAN-1/2004 (lane 4), and M2 protein of A/Thailand/1/KAN-1/2004 (lane 6) in 293T cells transfected by eukaryotic plasmid expression vectors. (B) Expression of rAd5 vectors was confirmed in A549 cells after transduction with vectors encoding HA (KAN-1) (lane 8), NP (KAN-1) (lane 9), and M2 (KAN-1) (lane 11). Arrows indicate the relevant predicted size of the indicated viral proteins. Bands refer to the right predicted size of different viral proteins that were detected in each lane as indicated. Molecular weight markers were used for protein size reference.</p

Humoral immune responses to HA, NP and M2 confirmed by ELISA after DNA/rAd5 immunization in ferrets.

<p>(A) Sera from the HA, HA+NP+M2 or vector control immunized ferrets were collected 14 days after the third DNA immunization (white bars), and 14 days after the recombinant adenovirus boost (solid bars), and subjected to ELISA assays to determine their end-point titer levels against HA(KAN-1), NP(KAN-1), and M2(KAN-1) antigens. Each bar represents the group mean for the end-point titers of total IgG and IgM, determined in duplicate by series dilution of ELISA assay with the error bars indicating the standard deviation. ANOVA tests were significant for only the responses against HA at the first time point, and for all three antigens after the rAd5 boost. For HA and M2, significant pairs of groups are noted on the graph. Only <i>p</i>-values less than 0.05 are indicated. * represents a <i>p</i>-value between 0.05 and 0.001, while ** indicates <0.001, and *** indicates <0.0001. As expected, the HA alone group elicited significant anti-HA immunity that increased after rAd5 HA boost (left panel) compared to controls (<i>p</i><0.0001). The HA+NP+M2 group elicited similar anti-HA ELISA antibodies, as well as significant anti-NP humoral responses (<i>p</i> = 0.0006) and anti-M2 responses (<i>p</i><0.0001) after the rAd boost (middle and right panels). (B) Sera from the NP, M2, NP+M2 or vector controls were collected 14 days after the third DNA immunization (white bars), and the sera from the same animals were also collected 14 days after the recombinant adenovirus boost (solid bars). ANOVA tests were not significant for any of the antigens at the first time point, and for NP and M2 after the rAd5 boost. For NP and M2, significant pairs of groups are noted on the graph. Only <i>p</i>-values less than 0.008 are indicated. * represents a <i>p</i>-value between 0.008 and 0.001, while ** indicates <0.001, and *** indicates <0.0001. For both NP and NP+M2 immunized groups, significant anti-NP humoral responses were observed after the rAd boost (<i>p</i><0.0001) (middle panel). Significant anti-M2 humoral responses were detected in animals immunized with NP+M2 post-rAd boost (<i>p</i> = 0.0005), but not in the M2 alone group (right panel).</p

Boost by adjuvanted RSV F DS-Cav1 can focus the RSV immune response to antigenic site Ø and II in elderly mice.

<p>Pre-immunized elderly mice (~85 weeks wait) were administered <b>(A)</b> DS-Cav1 formulated with SAS + Carbopol or <b>(B)</b> DS-Cav1 formulated with Alum and recognition of DS-Cav1, DS-Cav1 site Ø KO, DS-Cav1 site II KO, Post F and Post F site II KO probes by sera for all immunized mice assessed. Scatter plots show the geometric mean. <i>p</i> values were determined by two-tailed Wilcoxon matched-pairs signed rank test, <i>p</i> = >0.05 (ns); <i>p</i> < 0.05 (*). Associated raw data is reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186854#pone.0186854.s005" target="_blank">S5 Table</a>.</p

Adjuvants and their proposed mode of action.

<p>Adjuvants and their proposed mode of action.</p

Adjuvanted DS-Cav1 can augment RSV-neutralization titers in elderly mice.

<p><b>(A)</b> Pre-immunized elderly mice (DS-Cav1 adjuvanted with Poly (I:C), ~85 weeks wait) were administered DS-Cav1 formulated with Alum or SAS + Carbopol and the immune response for all mice assessed. <b>(B)</b> Neutralization titers for each group of seven elderly mice are shown. Scatter plots show the geometric mean (numerical value below), the palivizumab protective threshold is indicated by a dotted line [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186854#pone.0186854.ref010" target="_blank">10</a>]. <i>p</i> values were determined by two-tailed Wilcoxon matched-pairs signed rank test, <i>p =</i> >0.05 (ns); <i>p</i> < 0.05 (*). Associated raw data is reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186854#pone.0186854.s004" target="_blank">S4 Table</a>.</p

DS-Cav1-adjuvant formulations elicit antibodies of different IgG subclass in immunized mice.

<p><b>(A)</b> Sera from naïve mice primed and boosted with DS-Cav1 adjuvant mixtures were assayed by ELISA for their RSV F DS-Cav1 specific IgG1 and IgG2a antibody responses. Scatter plots show the geometric mean, closed circles and open triangles represent IgG1 and IgG2a antibody titers, respectively. (B) The ratios of IgG1 to IgG2a for each animal in the adjuvanted groups were calculated from the upper panel. <i>p</i>-values were determined using a two-tailed Mann-Whitney test and were adjusted for multiple comparisons using the Holm-Bonferroni method, <i>p</i> < 0.001 (***). Associated raw data is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186854#pone.0186854.s003" target="_blank">S3 Table</a>.</p

Prime-boost vaccination with different adjuvant formulations induced a balanced Th1/Th2 IgG response in elderly mice.

<p><b>(A</b>) Pre-immunized elderly mice (~85 weeks wait) were administered DS-Cav1 formulated with SAS + Carbopol or Alum. Sera were assayed by ELISA for their DS-Cav1 specific IgG1 and IgG2a antibody responses. Closed circles and open triangles represent IgG1 and IgG2a antibody titers, respectively. <b>(B</b>) The ratios of IgG1 to IgG2a for each animal in the groups were calculated from the upper panel. <i>p</i> values were determined by two-tailed Wilcoxon matched-pairs signed rank test, <i>p =</i> >0.05 (ns). Associated raw data is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186854#pone.0186854.s006" target="_blank">S6 Table</a>.</p