Evaluation of TB activity in immunized mouse sera by SMFA: one vaccine dose.

a<p>The proportion (percentage) of mosquitoes infected.</p>b<p>Median number of oocysts per mosquito (range).</p>c<p>% reduction = (mean control oocyst – mean test oocyst) ÷ mean control oocyst)*100.</p>d<p>Control.</p>*<p>CP – coat protein.</p

Pfs25-CP VLP purity and identity.

<p>(A) Deduced amino acid sequence of Pfs25-CP with Pfs25 sequence underlined. Amino acids boxed in the sequence were identified by N-terminal sequencing of the SDS-PAGE bands indicated. A Coomassie stain of an SDS-PAGE gel highlights the Pfs25-CP fusion polypeptide (arrowhead ‘a’) and CP monomer polypeptides (arrowheads ‘b’ & ‘c’). N-terminal sequencing of ‘a’ identified the first 5 amino acids of Pfs25, while sequencing of ‘b & c’ identified residue 26 of AlMV CP. (B) Western blot analysis of Pfs25-CP VLPs with an anti-Pfs25 mAb (left panel) and an anti-AlMV CP polyclonal serum (right panel).</p

Pfs25-CP VLP particle analysis.

<p>(A) Negative stain transmission electron micrograph of Pfs25-CP VLPs shows highly uniform particles of 19.3±2.4 nm in diameter. (B) Transmission electron micrograph of Pfs25-CP VLPs labeled with anti-Pfs25 and gold-labeled anti-mouse antibodies confirms the presence of Pfs25 on the particles. (C) DLS histogram showing a narrow size distribution for Pfs25-CP VLPs. The average hydrodynamic radius is ∼14 nm with a polydispersity of <15%. (D) Analytical SEC showing a single, major eluting species confirmed by Western blot analysis (not shown) to be Pfs25-CP VLP. The void volume of the SRT 1000 column (range 7.5 MDa –50 kDa) is indicated by (a) molecular weight standards indicated by (b) for thyroglobulin, (c) for BSA and (d) for uracil.</p

Schematic diagram of the ‘launch’ vector.

<p>Following agroinfiltration of plants, the sequence between the left border (LB) and the right border (RB) of the plasmid vector is transferred from <i>Agrobacteria</i> into plant cells where expression of the engineered TMV genome is driven by the Cauliflower mosaic virus (CaMV) 35S promoter. TMV replicase then drives amplification of primary transcript, and Pfs25-CP accumulation is then driven by the TMV CP subgenomic promoter (light blue box). Movement protein (MP) facilitates cell-to-cell transfer of viral sequences and is driven by the MP subgenomic promoter (dark blue box).</p

Anti-Pfs25 IgG responses in mice determined by ELISA.

<p>(A) Average anti-Pfs25 IgG titers from mice immunized with two doses of either 1.0 µg (circles) or 0.1 µg (squares) of Pfs25-CP VLPs, each with (open symbols) or without (filled symbols) Alhydrogel®, or 5.0 µg of CP only (black line). (B) Average anti-Pfs25 IgG titers from mice immunized with two doses of either 1.0 µg (circles), 0.1 µg (squares) or 0.01 µg (diamonds) of Pfs25-CP VLPs with Alhydrogel®. (C) Average anti-Pfs25 IgG titers elicited by a single administration of Pfs25-CP VLPs with Alhydrogel® at antigen doses ranging from 0.2–25 µg. Data are represented as average values per group of mice ± standard error of the mean.</p

Evaluation of TB activity in immunized mouse sera by SMFA: two vaccine doses.

a<p>The proportion (percentage) of mosquitoes infected.</p>b<p>Median number of oocysts per mosquito (range).</p>c<p>% reduction = (mean control oocyst – mean test oocyst) ÷ mean control oocyst)*100.</p>d<p>Control.</p>*<p>ns – not significant.</p>**<p>CP – coat protein.</p