Journal: bioRxiv

Article Title: Cryo-EM structure of endogenous Plasmodium falciparum Pfs230 and Pfs48/45 fertilization complex

doi: 10.1101/2025.02.13.638205

Figure Lengend Snippet: (A) Western blotting analyses using anti-Pfs230 LMIV230-01, anti-FLAG and anti-StrepII antibodies show that 230ΔD13D14 line expresses a truncated Pfs230, which runs at a lower molecular weight than the full-length protein present in 230FL. Both lines express C-terminal 3xFLAG and TwinStrepII tags. Anti-HSP70 was used as a loading control. All samples were run in non-reducing conditions. Molecular weight markers are shown on the left. (B) Immunofluorescence assay of stage V gametocytes from NF54/iGP2 expressing full-length Pfs230 or 230Δ1314. Fixed gametocytes were stained with anti-Pfs48/45 TB31F conjugated to Alexa 647 and anti- Pfs230 LMIV230-01 directly conjugated to Alexa 555. Parasite nuclei are stained with DAPI (cyan), merged and bright field (BF) images are shown. Scale bar = 5 μm. (C) Surface immunofluorescence assay (SIFA) of activated macrogametes (left panel) and microgametes (right panel) of NF54/iGP2 which express full length Pfs230 and two clonal lines of 230ΔD13D14. Gametes were stained with anti-Pfs48/45 TB31F conjugated to Alexa 647 and anti-Pfs230 LMIV230-01 directly conjugated to Alexa 555 prior to fixation. Merged and bright field (BF) images are shown. Scale bar = 5 μm. (D) Automated quantitation of Pfs230 localization observed in the SIFA of activated macrogametes, where anti-Pfs230 staining was classified as either being colocalized with anti-P48/45 staining (black) or absent while Pfs48/45 was staining was present (grey). The number of activated macrogametes counted for each parasite line is shown above each stacked bar graph.

Article Snippet: The slides were mounted with a coverslip using VECTASHIELD PLUS antifade mounting medium with DAPI (Vector Laboratories) and sealed with nail polish.

Techniques: Western Blot, Molecular Weight, Control, Immunofluorescence, Expressing, Staining, Quantitation Assay

Journal: bioRxiv

Article Title: Cryo-EM structure of endogenous Plasmodium falciparum Pfs230 and Pfs48/45 fertilization complex

doi: 10.1101/2025.02.13.638205

Figure Lengend Snippet: (A) Nanobody binding to Pfs230 D13D14 was evaluated by ELISA. Error bars represent standard deviation of the mean of two technical replicates. (B) Cladogram of the 100 most abundant nanobodies from Next-Generation Sequencing (NGS) after two rounds of phage display selection. Pfs230 D13D14 nanobodies identified by Sanger sequencing are labelled. Tree tips are scaled relative to abundance (counts per million). (C) Biolayer interferometry (BLI) affinity measurements using a two-fold dilution series of recombinant Pfs230 D13D14 from 6 – 200 nM binding to immobilized nanobodies. Measurements were plotted (solid line) and fitted to a 1:1 binding model (dashed line). Mean K D values for replicates with standard deviations are indicated, and representative binding curves are shown from two independent experiments. (D) Epitope binning competition experiments using BLI with the left column indicating immobilized nanobodies and the top row indicating nanobodies pre-incubated with Pfs230 D13D14 at a 10:1 molar ratio. Binding of Pfs230 D13D14 premixed with nanobody was calculated relative to Pfs230 D13D14 binding alone, which was arbitrarily assigned as 100%. The green and white boxes represent non-competition and competition, respectively. (E) Binding specificity of nanobodies to different Pfs230 domains and Pvs230 D13D14 by BLI. Representative curves of two independent experiments. (F) Immunofluorescence assay of stage V gametocytes using the indicated nanobody (Nb) detected with goat anti-alpaca IgG VHH domain conjugated to Alexa Fluor 488 (magenta) and anti-Pfs230 LMIV230-01 antibody conjugated to Alexa 555 (yellow). Parasite nuclei are stained with DAPI (cyan), merged and brightfield (BF) images are shown. Scale bar = 5 μm. (G) Pull down of Pfs230-Pfs48/45 complex from parasite lysate using StrepTactin XT resin in the presence of various antibodies or nanobody-Fcs. Presence of the Pfs230-Pfs48/45 complex was examined using western blotting under non- reducing conditions. Pfs48/45 and Pfs230 are indicated on the right. Representative blots of n = 3 independent experiments. I, input; U, unbound; E, eluate. Molecular weight ladder labeled on the left in kDa. (H) Transmission-blocking activity of anti-Pfs230 D13D14 nanobody-Fcs by standard membrane feeding assay. Number of oocysts per dissected midgut are plotted for three experiments. For experiments 1 and 2, nanobody-Fcs were added to blood meals at a concentration of 100 µg/mL. For experiment 3, nanobody-Fcs were added at a concentration of 200 µg/mL. LMIV230-01 was added at 200 µg/mL in all experiments. Bars show means with standard deviation. TRA, transmission reducing activity; N, number of uninfected mosquitoes / total number of mosquitoes.

Article Snippet: The slides were mounted with a coverslip using VECTASHIELD PLUS antifade mounting medium with DAPI (Vector Laboratories) and sealed with nail polish.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Next-Generation Sequencing, Selection, Sequencing, Recombinant, Incubation, Immunofluorescence, Staining, Western Blot, Molecular Weight, Labeling, Transmission Assay, Blocking Assay, Activity Assay, Membrane, Feeding Assay, Concentration Assay

Journal: bioRxiv

Article Title: Cryo-EM structure of endogenous Plasmodium falciparum Pfs230 and Pfs48/45 fertilization complex

doi: 10.1101/2025.02.13.638205

Figure Lengend Snippet: (A) Schematic of full-length Pfs230 with the recombinant fragments to D1D2, D5D6, D11D12 and D13D14 indicated. Signal peptide is labeled as SP. (B) Multiplex assay for reactivity of plasma against Pfs230 and Pvs230 domains from PNG individuals with current P. falciparum infection (Pf pos) as compared to those who had a prior infection (Pf prior) or naïve individuals from Melbourne. The dashed line represents an antigen-specific seropositivity cut-off, calculated as the mean of the negative control samples plus two times the standard deviation. Statistical significance between groups was calculated using a Kruskal-Wallis test, with Dunn’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (C) Groups of C57BL/6 mice were immunized with 10 μg of either 230D13D14L or 230D13D14S mRNA-LNP alone. Mice were immunized (IM) on day 1 and day 28. For pre-immune sera, mice were bled on day 1 prior to immunization. Mice were then bled on day 21 and day 68 for the first (B1) and second (B2) bleed. Pfs230 D13D14 antibody levels were measured by endpoint dilution ELISA. Error bars represent 95% confidence intervals of the geometric mean. n = 6 mice per immunization group. (D) Western blot analyses showing immunized mouse sera are specific to Pfs230 D13D14 in sexual stage parasites. Anti- HSP70 was used as a loading control. Molecular weight markers are shown on the left. (E) Immunofluorescence assay of stage V gametocytes from NF54/iGP2. Fixed gametocytes were stained with pre-immune or second bleed pooled sera from mice immunized with 230D13D14S mRNA-LNP. (F) Surface immunofluorescence assay (SIFA) of activated macrogametes from NF54/iGP2. Macrogametes were stained with pre-immune or second bleed pooled sera from mice immunized with 230D13D14S mRNA-LNP. For panels E and F, mouse sera were detected using donkey anti-mouse conjugated to Alexa 647 and anti- Pfs230 LMIV230-01 was directly conjugated to Alexa 555. Parasite nuclei are stained with DAPI (cyan), merged and bright field (BF) images are shown. Scale bar = 5 μm.

Article Snippet: The slides were mounted with a coverslip using VECTASHIELD PLUS antifade mounting medium with DAPI (Vector Laboratories) and sealed with nail polish.

Techniques: Recombinant, Labeling, Multiplex Assay, Infection, Negative Control, Standard Deviation, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Molecular Weight, Immunofluorescence, Staining