Total IgG antibody titer correlates with oocyst number.

<p>BALB/c mice (n = 10) were immunized with Ad-MVA Pfs25 and infected with Pfs25DR3 as previously (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029428#pone-0029428-g004" target="_blank">figures 4</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029428#pone-0029428-g005" target="_blank">5</a>). Mean oocyst number (circles) and the prevalence of infection (squares) were calculated per mouse as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029428#pone-0029428-g004" target="_blank">figures 4a</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029428#pone-0029428-g005" target="_blank">5a</a>. These data are shown plotted against specific anti-Pfs25 total IgG titer for each mouse.</p

Parasite infectivity (per mosquito) following Ad-MVA Pfs25 immunization.

<p>BALB/c mice were immunized with Ad-MVA Pfs25 (red) as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029428#pone-0029428-g001" target="_blank">figure 1a</a> or Ad-MVA GFP (blue) a GFP-encoding control. Mice were divided into two groups and infected with (<i>A</i>) Pfs25DR3 (10 per group) or (<i>B</i>) wild-type <i>P. berghei</i> (5 per group) and used to assess transmission to mosquitoes. Mosquito midguts were examined 12 days post-feeding. Data points represent mosquitoes that fed on individual mice. X-axis points represent individual mice. Horizontal lines indicate the mean number of oocysts for each mouse (+/− sem).</p

Parasite prevalence (per mouse) following Ad-MVA Pfs25 immunization.

<p>The same mosquitoes from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029428#pone-0029428-g004" target="_blank">figure 4</a> were analyzed for oocyst prevalence. Data points represent the prevalence of oocyst infection in mosquitoes that fed on individual mice, expressed as a percentage of total mosquitoes dissected.</p

Pfs25 specific total IgG responses following Ad-MVA Pfs25 and ChAd63 Pfs25 immunization.

<p><i>(A)</i> BALB/c mice were immunized with Ad-MVA Pfs25 (AdHu5 Pfs25 prime, MVA Pfs25 boost). Total IgG responses against recombinant Pfs25 protein were measured by ELISA in the serum of mice taken at the number of weeks following first immunization as shown. Mean ELISA titers ± sem (n = 10) are shown. Titers increased over time following the first immunization and were boosted following MVA Pfs25 administration at week 10 (arrow). The dotted line represents the titer required to provide 50% efficacy against <i>P. falciparum</i> in a SMFA using alternative vaccine platforms, according to previously published methods. <i>(B)</i> BALB/c mice were immunized with ChAd63 Pfs25 (n = 25) or AdHu5 Pfs25 (n = 10). Total IgG responses against recombinant Pfs25 protein were measured by ELISA in the serum of mice taken 8 weeks following a single immunization. Geomean ELISA titers ±95% CI are shown.</p

Pfs25 specific IgG isotype responses following Ad-MVA Pfs25 immunization.

<p>BALB/c mice were immunized with Ad-MVA Pfs25 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029428#pone-0029428-g001" target="_blank">figure 1a</a>. Mice were immunized in two separate experiments and the data show pooled responses (n = 17–20). Individual isotype IgG responses against recombinant Pfs25 protein were measured by ELISA in the serum of mice taken at 2 weeks following final immunization. Individual and mean OD results are shown for samples measured at a dilution of 1 in 500. Positive (immune) and negative (non-immune) control sera were included in all assays. IgG3 was undetectable at this dilution.</p

Parasite infectivity via membrane feeding asssay (per mosquito) following Ad-MVA Pfs25 immunization.

<p>Sexually mature <i>P. falciparum</i> NF54 cultures were mixed with anti-Pfs25 serum from a representative Ad-MVA immunized mouse (Pfs25) or negative control anti-GFP serum (GFP) at final dilutions of 1 in 5, 1 in 10 and 1 in 100. Data points represent the number of oocysts found in individual mosquitoes 12 days post feed. Horizontal lines indicate the mean number of oocysts (+/−sem).</p

Generation and phenotypic analysis of Pfs25DR3.

<p><u>(A) Schematic representation of the construction of Pfs25DR3.</u> The targeting plasmid was linearized by restriction digestion, and DNA was transfected into <i>P. berghei</i> ANKA 2.34. The <i>P. berghei</i> ORFs P25 and P28 were replaced with the <i>P. falciparum</i> gene sequence of Pfs25 and the selectable marker cassette (TgDHFR/TS). (<i>B</i>) <u>Diagnostic PCR on cloned transgenic Pfs25DR3</u>. (M: 500 bp marker; 1: 5′ integration, oligos Int1+Int2; 2: 3′ integration, oligos Int3+Int4; 3: Pf25 presence, oligos Pf25F+Pf25R; 4: Pb25 presence, oligos Pb25F+Pb25R. (C) <u>Western blot analysis of Pfs25DR3 ookinetes</u>. Purified ookinetes (5×10⁷ per lane) were hybridized with anti-Pfs25/GFP serum from Ad-MVA immunized mice. Loading control was carried out on stripped nitrocellulose membrane with anti-TAT-1 antibody at 1∶10,000 (primary), and anti-mouse IgG at 1∶10,000 (secondary). Lane 1: <i>P. berghei</i> WT 2.34 ookinetes; Lane 2: <i>P. berghei</i> Pfs25DR3 ookinetes. (D) <u>Expression and localization of Pfs25 in the Pfs25DR3 transgenic parasite</u> was confirmed via immunofluoresence assay on PFA fixed ookinetes.</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

Multi-functionality of CD4⁺ T cell response induced by vaccination.

<p>The multi-functional composition of the CD4⁺ T cell response (measured in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020977#pone-0020977-g008" target="_blank">Figure 8</a>) is shown. The pie charts summarize the fractions of AMA1-specific CD4⁺ cells that are positive for a given number of functions (IFN-γ, TNF-α and IL-2). Individual data points and median percentage of the parent CD4⁺ T cell response (open bars) are shown for each of the functional populations indicated on the x-axis.</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