Additional file 1: of The comparative diagnostic accuracy of the Mini Mental State Examination (MMSE) and the General Practitioner assessment of Cognition (GPCOG) for identifying dementia in primary care: a systematic review protocol

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Adjuvanticity of IMX313 in rabbits following immunization with AdHu5-PfAMA1.

<p>New Zealand white rabbits were immunized i.m. with 1×10⁹ or 5×10⁷ ifu AdHu5-PfAMA1±IMX313, 56 days later they were given a homologous boost. Serum was taken on days 0, 14, 55, 70 and 84 and IgG titers were measured by ELISA (A). Median titer and range for n = 4 rabbits/group are shown. The arrows indicate the times of immunization. Purified IgG from day 84 post-immunization was assayed for GIA (B). Data presented are percentage inhibition for individual rabbits relative to the parasite growth observed without sera using purified IgG at 2.5 mg/mL. The non-linear regression curve is shown. Data show the results of a single experiment.</p

Adjuvant activity of IMX108 following immunization with AdHu5-PyMSP1₄₂.

<p>BALB/c mice were immunized i.m. with 1.5×10⁹ or 1.5×10⁷ ifu AdHu5-PyMSP1₄₂±IMX108 (Ad42-IMX108 or Ad42-Nil). Fourteen days post immunization splenocytes were re-stimulated with PyMSP1₃₃ peptides and assessed for frequency of IFN-γ, IL-2, and TNFα positive CD8⁺ (A) and CD4⁺ (B) T cells by ICS. Box and whisker plots show median, IQR and range. The pie charts show the functional composition of the response and represent the proportion of PyMSP1₃₃⁻specific CD8⁺ T cells positive for 1, 2 or all 3 cytokines measured. Fourteen days post-immunization total PyMSP1₁₉-specific IgG responses were measured by ELISA (C). Data points represent individual mice and bars indicate median titer. n = 5 mice per group. **<i>P</i><0.01, *<i>P</i><0.05 compared to AdHu5-PyMSP1₄₂+IMX108 at the same dose by Mann Whitney test. Data are representative of two independent experiments.</p

Adjuvanticity of IMX108 in FcRγ^−/− mice.

<p>Wildtype or FcRγ^−/− mice were immunized i.m. with 1.5×10⁹ ifu Ad42-IMX108 or Ad42-Nil. Two weeks later PyMSP1₃₃-specific IFN-γ, TNFα and IL-2 positive CD8⁺ T cells in the spleen were measured by ICS (A). Box and whisker plots show median, IQR and range for n = 7–13 mice/group. PyMSP1₁₉-specific total IgG was measured by ELISA (B). Points indicate individual mice and bars indicate median titer. n = 6 mice/group. *<i>P</i><0.05, **<i>P</i><0.01, by Mann Whitney test. Combined data are shown from two independent experiments.</p

Schematic representation of PfAMA1±IMX313 and PfM128±IMX313 and assessment of antigen oligomerization by western blotting.

<p>Schematic representation of PfAMA1±IMX313 (A) and PfM128±IMX313 (B) constructs are shown; human tissue plasminogen activator signal peptide (tPA) (30 αα), PfAMA1 (521 αα), PfM128 (1111 αα) and IMX313 (55 αα). PfAMA1 3D7 protein was included as a positive control. Western blots probed with mouse anti-PfAMA1 polyclonal antibody (C) and mouse anti-PfMSP119 polyclonal antibody (D) under both reducing and non-reducing conditions. To the left of each column, * indicates presumed dimers and # indicates presumed multimers. Control AdHu5 infected lysate without the antigen of interest was also included and no bands were observed (data not shown).</p

Adjuvant activity of IMX108/IMX313 in homologous prime-boost regimes.

<p>Frequency of antigen-specific IFN-γ, TNFα and IL-2 positive CD4⁺ and CD8⁺ T cells in the spleen were measured by ICS fourteen days post-boost following two immunizations with 5×10¹⁰vp AdHu5-PyMSP1₄₂±IMX108 i.d. (Ad-Ad42+IMX108 and Ad-Ad42-Nil respectively) (A), 5×10⁷ pfu MVA-PyMSP1₄₂±IMX108 i.d. (M-M42+IMX108 and M-M42-Nil) (B) or 1×10⁹ ifu AdHu5-PfAMA1±IMX313 i.m. (Ad-AdAMA1+IMX313 and Ad-AdAMA1-Nil) (C). Immunizations were given eight weeks apart. Box and whisker plots show median, IQR and range and pies show the proportion of cells positive for 1, 2 or all 3 cytokines. Antigen-specific total IgG responses were also measured in the serum fourteen days after boost by ELISA (D-F). Points represent individual mice and bars indicate median titer. The dashed line indicates the limit of detection. **<i>P</i><0.01, *<i>P</i><0.05 compared to the same vaccination regime with IMX108 or IMX313 by Mann Whitney test. Data show the results of single experiments.</p

Adjuvant activity of IMX313 following immunization with AdHu5-PfAMA1 or AdHu5-PfM128.

<p>BALB/c mice were immunized i.m. with 1×10⁹ ifu AdHu5-PfAMA1±IMX313 (AdAMA1-IMX313 and AdAMA1-Nil). Twenty-one days post-immunization frequency of IFN-γ, TNFα and IL-2 positive CD8⁺ and CD4⁺ T cells was assessed in the blood by ICS following stimulation with PfAMA1 peptides (A). Antigen-specific total IgG was assessed by ELISA (B). Points represent individual mice and bars indicate median responses (n = 5–6 mice per group). **<i>P</i><0.01, *<i>P</i><0.05 compared to AdAMA1-IMX313 by Mann Whitney test. Data show the results of a single experiment. A similar result was observed by spleen IFN-γ ELISPOT (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044943#pone.0044943.s003" target="_blank">Figure S3</a>). In a separate experiment, mice were immunized i.m. with 1×10⁷ ifu AdHu5-PfM128±IMX313 (AdPfM128-IMX313 and AdPfM128-Nil). Fourteen days later mice were culled and PfMSP1₃₃-specific IFN-γ positive splenocytes were measured by ELISPOT (C). Data shown are spot forming units (SFU) per 10⁶ splenocytes. PfMSP1₁₉-specific total IgG was assessed by ELISA (D). Bars indicate median responses (n = 5 mice per group). Box and whisker plots indicate median, IQR and range. The dashed line indicates the limit of detection. ⁺<i>P</i><0.05 compared to AdPfM128-IMX313 by Mann Whitney test.</p