Alum-adjuvanted gH1-Qβ performs as well as Panvax in mice vaccinated and then challenged with the 2009 pandemic influenza strain.

<p>Groups of five BALB/c mice were immunized subcutaneously at days 0 and 28 with indicated doses of gH1-Qβ (either alone or adjuvanted with alum) or Panvax. Shown are the means (and standard errors) of serum hemagglutination inhibition (HAI) titers against pH1N1 2 weeks post second dose (A), virus micro-neutralization titers against pH1N1 2 weeks post second dose (B) and virus lung titers following challenge with pH1N1 3 weeks post second dose (C). One way ANOVA on the log transformed titers was used to compare groups and the significance level set at 5%. All ANOVA tests were found to be significant and were followed up with Tukey’s honest significant difference test to compare between any 2 groups. Significant differences between vaccine response and PBS control are outlined with *, p<0.05. Reported p values are adjusted to account for multiple comparisons. The abbreviation, ns, indicates no significant difference between the indicated groups.</p

Alum-adjuvanted gH1-Qβ induces broad gH1 and Qβ specific T cell responses characterized by the production of IFNγ.

<p>HA and Qβ specific T cell responses were measured in pooled splenocytes from three BALB/c mice by secreted cytokine concentration after stimulation with peptide pools spanning the HA and Qβ regions of the gH1-Qβ vaccine. Mice were immunized with 10 µg alum-adjuvanted gH1-Qβ and 1.5 µg of Panvax. Each bar represents the total cytokine response to each peptide pool and each box the response to each cytokine. Names designated to each peptide pool are indicated on the x axes. Each data point is the response from splenocytes pooled from three mice.</p

The presence of ssRNA in gH1-Qβ VLPs is responsible for an increased IgG2a bias in the influenza-specific antibody repertoire.

<p>Groups of five BALB/c mice were immunized subcutaneously at days 0 and 28 with 4 µg gH1, 10 µg gH1-Qβ(pGlu) or 10 µg gH1-Qβ, either alone or adjuvanted with Alum. Antibody responses were assayed 2 weeks post second dose. (A) Mean (and standard error) of virus neutralization titers against pH1N1. One way ANOVA on the log transformed titers was used to compare groups and the significance level set at 5%. The ANOVA test was significant and Tukey’s honest significant difference test was then used to compare between any 2 groups. Significant differences between vaccine response and PBS control are outlined with *, p<0.05. (B) Mean (and standard error) of gH1-specific IgG1 and IgG2a antibody isotype responses determined by ELISA. Responses are also shown as ratios of gH1-specific IgG2a:IgG1 isotype geometric mean titers, with titers defined as those dilutions that reached half the maximal OD observed for the assay.</p

Alum-adjuvanted gH1-Qβ elicits protective immunity in Ferrets.

<p>Ferrets were immunized twice intramuscularly on days 0 and 21 with 100 µg of non-adjuvanted gH1-Qβ, 100 µg alum-adjuvanted gH1-Qβ or 15 µg Panvax. (A) Geometric mean (and 95% confidence intervals) for neutralizing antibody responses. A two-way repeated-measures ANOVA was performed on the log transformed titers with time point and vaccine as the factors. Both factors and their interaction were found to be significant at the 5% significance level and Bonferoni’s corrected multiple comparison test used to compare between 2 groups. Significant differences between vaccine response and PBS control are outlined with *, p<0.05. (B) Mean (and standard error) of activity and respiratory scores (0 indicating no changes, 1 indicating reduced activity and mild respiratory signs, and 2 representing depressed behavior and moderate respiratory signs), body weight and body temperature measured over the 6 days following challenge with pH1N1. Clinical scores and body weight for both the alum-adjuvanted and non-adjuvanted gH1-Qβ and Panvax were not significant from each other (p>0.05), but significantly different from the control PBS group as determined by linear mixed model (p<0.05). (C) Mean (and standard error) of viral titers in nasal washes at indicated days after challenge with pH1N1. Titers for all vaccines were significantly different from the control PBS group as determined by linear mixed model (p<0.01).</p

The presence of ssRNA in Qβ VLPs is responsible for an increased Th1 bias in the influenza-specific CD4+ T cell response.

<p>Mice were immunized with 1 µg gH1, 10 µg gH1-Qβ or 10 µg gH1-Qβ(pGlu) as indicated, either non-adjuvanted or adjuvanted with alum, and T cell responses measured two weeks post second dose. (A) Frequency of HA and Qβ specific IFNγ-producing CD4⁺ T cells was determined by intracellular staining after stimulation with peptides from the HA (peptide pools HA 4, HA5, HA6, HA8 & HA9) and Qβ (peptide pools Qβ 2, Qβ 3, Qβ 4 & Qβ 6) regions of the gH1-Qβ vaccine. Each data point is the total response obtained by adding together individual responses measured for each peptide pool. Mouse splenocytes were pooled from at least three mice and data are representative of at least two experiments performed with similar results. Unstimulated samples were subtracted from the peptide-stimulated samples. (B) Plot of the relative proportion of CD4⁺ and CD8⁺ contributions to IFNγ⁺ T cell responses, as obtained in (A), to ex-vivo stimulation of HA and Qβ peptides for mice immunized with 10 µg alum-adjuvanted gH1-Qβ. (C) Histogram showing the frequency of CD4⁺ T cells, determined by intracellular labeling, that are positive for the indicated cytokine following ex-vivo stimulation with recombinant gH1 or diluent alone. Pie-charts display responses plotted as relative frequency of IFNγ, IL-4 and IL-13 producing CD4⁺ T cells, with values from unstimulated samples subtracted from the Ag-stimulated sample. Each data point is the mean response of three replicate measurements from splenocytes pooled from at least three mice and is representative of at least two experiments performed with similar results. (D) Total secreted cytokine levels to the indicated HA and Qβ peptide pools. Each data point is the response from splenocytes pooled from at least three mice and is the mean of two independent experiments (error bars represent the range).</p