Recognition of native Pfs48/45 in <i>P. falciparum</i> gametocyte extract.

<p>(a) Western blot analysis with non-reduced (left panel) or reduced (right panel) <i>P. falciparum</i> gametocyte extract against serum of individual mouse immunized with either CFA or ISA-51 or alum formulation. Stage V gametocyte extract was run either in non-reduced or reduced (10 mM 2-mercaptothanol) form in SDS-PAGE and transferred to nitrocellulose membrane. Mice sera were allowed to react at 1∶1000 dilution for 1 h at 22°C. HRP-conjugated anti-mouse IgG at 1∶10000 dilution was used as detection antibody and was developed using ECL substrate. Lane 1, mAb IIC5B10; lane 2, one representative mouse serum immunized in CFA; lane 3, one representative mouse serum immunized in Montanide ISA-51; lane 4, one representative mouse serum immunized in alum formulation. The figure is assembled from separate experiments. (b) Mouse sera (1∶1000 dilution) were tested by live immunofluorescence assays as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006352#s4" target="_blank">materials and methods</a>.</p

Follow up of immune responses elicited by CH-rPfs48/45 in baboons.

<p>Analysis of anti-CH-rPfs48/45 IgG titers (open bars) and percent transmission blocking activity (closed bars) upto 7 months post second boost in baboons. Results show mean antibody titer and mean transmission blocking activity of 5 baboons +95% CI.</p

Transmission blocking activity of sera of different bleeds at various time points from baboons immunized with CH-rPfs48/45 in Montanide ISA-51.

*<p><i>P</i><0.0001 (Mann-Whitney test).</p><p>MFA was done with sera collected on 12/10/2007 (1 mo post primary immunization), 01/10/2008 (1 mo post 1^st boost, 02/21/2008 (15 d post 2^nd boost), 03/06/2008 (1 mo post 2^nd boost), and 05/05/2008 (3 mo post 2^nd boost). The geometric mean of oocyst numbers/midgut in the presence of individual pre-immune sera were 22.24 (Pan 3104), 18.43 (Pan 3140), 22.1 (Pan 3163), 6.31 (Pan 3275), and 19.7 (Pan 3313), respectively. Numbers within parenthesis represent total number of infected mosquitoes/total number of mosquitoes dissected for each feed.</p

Analysis of anti-Pfs48/45 antibody production by Olive baboons (<i>Papio anubis</i>).

<p>(a) Each animal was immunized with CH-rPfs48/45 (50 µg in 0.25 ml endotoxin free PBS) formulated in Montanide ISA-51 (0.25 ml) in baboons, administered intra muscularly (quadriceps, two sites). Schedules for immunization and bleeds are indicated and sera were stored at −20°C until shipped frozen from Kenya to Baltimore for ELISA and MFA. The samples were shipped under an export permit CITES # 008101. (b) Anti-Pfs48/45 whole IgG titer at various time points analyzed by ELISA. Pre-immune +3 x SD is shown by solid horizontal lines. ELISA readings with sera dilutions, 1 month post primary immunization (filled square), 1 month post first boost (filled triangle) and 1 month post second boost (filled diamond) are shown with±SD for individual baboons (Pan 3104, Pan 3140, Pan 3163, Pan 3275, Pan 3313). (c) Distribution of anti-Pfs48/45 IgG1(solid diamonds) and IgG2 (open diamonds) subtypes in 1 month post primary immunization sera (Dec 10), 1 month post 1^st boost (Jan 10), 1 month post 2^nd boost (Mar 06), and 3 months post 2^nd boost (May 05). Data are presented as mean OD₄₀₅ value±SD for individual baboon.</p

Figure 2

<p>(a) ELISA analysis of CH-rPfs48/45 immunized individual mouse sera in three different adjuvant formulations: Complete Freund's adjuvant (top panel), Montanide ISA-51 (middle panel), and Alum (bottom panel). All the results are representative of three independent experiments. Pooled pre-immune sera + 3SD are shown by broken lines. ELISA OD₄₀₅ values for individual mice are shown; mouse 1 (filled triangle), mouse 2 (open square), mouse 3 (filled square), mouse 4 (open circle), mouse 5 (filled circle). (b) Analysis of anti-Pfs48/45 IgG isotype distribution in individual mouse sera: IgG1 (filled columns), IgG2a (hatched columns), IgG2b (stippled columns), IgG3 (blank columns).</p

MFA with sera from mice immunized with CH-rPfs48/45 formulated in CFA, Montanide ISA-51 or Alum.

<p>Individual mouse sera were tested for transmission blocking activity with respect to corresponding pooled pre-immune sera. Data are represented as percent transmission blocking activity (reduction in the number of oocysts per mosquito midgut). Numbers within parenthesis represent total number of infected mosquitoes/total number of mosquitoes dissected for each feed.</p