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 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

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

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

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 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

Assessment of antibody isotype profiles following vaccination and CHMI. Isotype profiles of serum antibody responses were assessed by ELISA.

<p>(A) Anti-MSP1₁₉ responses in the VAC037 Phase Ia clinical trial (open symbols) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy1" target="_blank">[19]</a> and the VAC039 Phase IIa clinical trial (closed symbols) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a>. Responses are shown at baseline (d0); following ChAd63 MSP1 priming immunization (d28); following the MVA MSP1 boost (d84 in the Phase Ia trial or dC−1 in the Phase IIa trial); or following CHMI at day of diagnosis, dC+DoD, or at first follow-up post drug treatment, dC+35. (B) Anti-AMA1 responses in the VAC036 Phase Ia clinical trial <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy2" target="_blank">[20]</a> and VAC039 Phase IIa trial <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a>, reported as in panel A. In all panels, individual and median responses are shown, n = 4<b>–</b>13 depending on sample availability for each tested time-point.</p

Associations between IgG antibody responses and CHMI outcome measures.

<p>Associations are reported between anti-MSP1₁₉ or anti-AMA1 total IgG ELISA titer readouts at various time-points and/or fold-change post-CHMI (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone-0107903-g001" target="_blank">Figure 1E</a>), as well as with time to malaria diagnosis by thick-film microscopy during CHMI, and parasitemia at time of diagnosis (measured by qPCR in terms of parasites/mL blood). In all cases, Spearman’s rank correlation coefficient and <i>P</i> value are shown. n/a = not applicable; n.d. = not done. *For these analyses, relevant vaccine groups were included: for the MSP1₁₉ analysis, data were combined from the MSP1-only vaccination group and from the MSP1+AMA1 and MSP1+ME-TRAP co-administration groups; and for the AMA1 analysis data were combined from the AMA1-only vaccination group and from the MSP1+AMA1 co-administration group.</p><p>Associations between IgG antibody responses and CHMI outcome measures.</p

Assessment of antibody isotype profiles following vaccination, CHMI and natural exposure.

<p>Isotype profiles of serum antibody responses were assessed by ELISA. (A) Individual and median anti-MSP119 serum antibody isotype responses are shown for MSP1-only vaccinees at the peak after the MVA MSP1 boost (“Vaccine”, <i>n</i> = 12) and at dC+35 following CHMI (“Vaccine+CHMI”, <i>n</i> = 11) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a>; at dC+35 for 18 infectivity control volunteers from three separate CHMI studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Ewer1" target="_blank">[22]</a>; and from 20 naturally-exposed immune adults from Kilifi, Kenya. (B) Individual and median anti-AMA1 serum antibody isotype responses are shown for AMA1-only vaccinees at the peak after the MVA AMA1 boost (<i>n</i> = 9) and at dC+35 following CHMI (<i>n</i> = 9) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone.0107903-Sheehy3" target="_blank">[21]</a>; and for infectivity control volunteers and naturally-exposed immune adults from Kilifi, Kenya as in panel A.</p

Associations between IgG antibody avidity and total IgG ELISA titer.

<p>Associations are reported between anti-MSP1₁₉ ETSR allele or anti-AMA1 3D7 allele total IgG ELISA titer readouts (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone-0107903-g001" target="_blank">Figure 1B, D</a>) and the corresponding IgG avidity measurement by NaSCN-displacement ELISA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone-0107903-g003" target="_blank">Figure 3E, F</a>). Groups were assessed according to vaccination and/or parasite exposure status as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107903#pone-0107903-g003" target="_blank">Figure 3E, F</a>. In all cases, Spearman’s rank correlation coefficient and <i>P</i> value are shown. n.d. = not done (too few positive responders). *Data from vaccinees receiving only a single vaccine (i.e. MSP1-only or AMA1-only) were used for this analysis.</p><p>Associations between IgG antibody avidity and total IgG ELISA titer.</p