Cellular and humoral responses following different liposome + OVA formulations.

<p>The DDA:TDB+OVA formulations indicated along the X-axis were given to C57/BL6 mice as three homologous injections, two weeks apart. Splenocytes were isolated at the final timepoint (14 weeks) and responses to a CD8 OVA epitope (A) and two CD4 epitopes (B, C) were assessed by <i>ex vivo</i> IFNγ ELISpot. D) Total IgG antibody responses to whole OVA protein at the final time-point, as measured by an end-point titre ELISA. *p<0.05, ** p<0.01.</p

Liposome specifications of the DDA:TDB formulations containing OVA and TLR agonists.

<p>Liposome specifications of the DDA:TDB formulations containing OVA and TLR agonists.</p

Cellular and humoral responses to liposome-adjuvanted viral vectored vaccines.

<p>T cell and antibody responses were assessed in Balb/c mice at peak time-points for Adenovirus (Ad) and Modified Vaccinia Ankara (MVA) viral vectored vaccines expressing TIPeGFP, i.e. two weeks after a single injection of Ad and one week after two injections of MVA, combined with cationic DDA:TDB or neutral DSPC/TDB liposomes. Spleen T cell responses to two CD8 (A, B) and one CD4 epitope (C), contained within the insert of the viral constructs, were measured using IFNγ ELISpot. D) Total IgG titre in the serum of mice immunised with the Ad+liposome formulations at the peak time-point. **p<0.01.</p

Size distribution (bars) and Z-potential (points) of DDA:TDB liposomal formulations with OVA±pIC/CpG.

<p>Size distribution (bars) and Z-potential (points) of DDA:TDB liposomal formulations with OVA±pIC/CpG.</p

IFN-γ, IL-2, TNF and CD107a responses to the vaccine insert measured by flow cytometry.

<p>Production of IFN-γ (A), IL-2 (B) and TNF (C), and mobilization of CD107a (D), after background subtraction in CD3⁺CD4⁺ (black circles) and CD3⁺CD8⁺ (white circles) cell populations stimulated with a single pool of peptides spanning the complete NP+M1 vaccine insert. Volunteers in group 3 were tested at weeks 0, 1, and 3. Significant differences between pre- and post-vaccination time points were detected using the Wilcoxon signed rank test as follows: IFN-γ CD4⁺, week 1 (p = 0·0001) and week 3 (p = 0·0001); IFN-γ CD8⁺, week 1 (p = 0·001) and week 3 (p = 0·0005); IL-2 CD4⁺, week 1 (p = 0.001) and week 3 (p = 0·0001); IL-2 CD8⁺, week 1 (p = 0·006) and week 3 (p = 0·03); TNF CD4⁺, week 1 (p = 0·002) and week 3 (p = 0·0003); TNF CD8⁺, week 1 (p = 0·0003) and week 3 (p = 0·002); CD107a CD8⁺, week 3 (p = 0·004).</p

Phenotypic and clonotypic properties of M1-specific CD8⁺ T cells elicited by MVA-NP+M1.

<p>(A) Phenotype of vaccine-elicited CD8⁺ T cells specific for the HLA A*0201-restricted M1-derived epitope GILGFVFTL (residues 58–66). Antigen-specific CD3⁺CD8⁺tetramer⁺ cells are shown as coloured dots superimposed on bivariate plots showing the phenotypic distribution of the total CD8⁺ T cell population (grey density plots). Response sizes were 1·48% (left panels) and 0·75% (right panels) with respect to the total CD8⁺ T cell population. (B) TRBV and TRBJ usage, CDR3 amino acid sequence and relative frequency of the GILGFVFTL-specific CD8⁺ T cell clonotypes contained within the antigen-specific populations depicted in (A). Public clonotypes within the present dataset are colour-coded. Representative analyses are shown for volunteers in group 3 (70+ years).</p

Ex vivo IFN-γ ELISpot responses to the vaccine insert.

<p>(A) Median and individual ex vivo IFN-γ ELISpot responses from vaccinated volunteers at baseline (week 0), and weeks 1, 3, 8, 12, 24, and 52. Significant differences between the pre- and post-vaccination time points were detected using the Wilcoxon signed rank test: week 1 (p = 0·0001), week 3 (p = 0·0001), week 8 (p = 0·0001), and week 12 (p = 0·001). (B) Median <i>ex vivo</i> IFN-γ ELISpot responses to the NP+M1 insert stratified according to age: black bars  =  group 1 (50–59 years), white bars  =  group 2 (60–69 years), and grey bars  =  group 3 (70+ years). Error bars indicate interquartile ranges. Significant differences between the pre- and post-vaccination time points were detected using the Wilcoxon signed rank test as follows. Group 1: week 1 (p = 0·002), week 3 (p = 0·002), week 8 (p = 0·002), week 12 (p = 0·039), week 24 (p = 0·002), and week 52 (p = 0·0039). Group 2: week 1 (p = 0·002), week 3 (p = 0·002), week 8 (p = 0·002), and week 12 (p = 0·0371). Group 3: week 1 (p = 0·0039) and week 3 (p = 0·0195). Significant differences were also detected between groups using the Mann-Whitney U-test, with responses in group 1 being higher than those in group 3 at week 3 (p = 0·043) and week 8 (p = 0·023). (C) Median and individual <i>ex vivo</i> IFN-γ ELISpot responses at week 1 and week 3 stratified according to age, and including a vaccinated cohort of younger (18–45 years) volunteers.</p

Demographic characteristics of volunteers vaccinated in each cohort.

<p>Demographic characteristics of volunteers vaccinated in each cohort.</p

Frequency of local and systemic adverse events that were possibly, probably or definitely related to vaccination.

<p>(A) Volunteers aged 50+ (n = 30). (B) Volunteers aged 18–45 (n = 15). For both age groups pain was the most frequently recorded local adverse event followed by erythema. A similar pattern of systemic adverse events was observed in both age groups with the majority of solicited adverse events occurring in 20–60% of individuals. For volunteers aged 18–45, 85% of adverse events were mild; for volunteers aged 50+, 87% of adverse events were mild.</p

Patterns of clonotype usage in M1-specific CD8⁺ T cell populations before and after vaccination with MVA-NP+M1.

<p>TRBV and TRBJ usage, CDR3 amino acid sequence and relative frequency are shown for GILGFVFTL-specific CD8⁺ T cell clonotypes on day 0 (pre-vaccination) and day 7 (post-vaccination). Public clonotypes within the present dataset are colour-coded. Non-public clonotypes present at both time points within an individual are highlighted in bold type.</p