Robust, reproducible, industrialized, standard membrane feeding assay for assessing the transmission blocking activity of vaccines and drugs against Plasmodium falciparum.

BackgroundA vaccine that interrupts malaria transmission (VIMT) would be a valuable tool for malaria control and elimination. One VIMT approach is to identify sexual erythrocytic and mosquito stage antigens of the malaria parasite that induce immune responses targeted at disrupting parasite development in the mosquito. The standard Plasmodium falciparum membrane-feeding assay (SMFA) is used to assess transmission-blocking activity (TBA) of antibodies against candidate immunogens and of drugs targeting the mosquito stages. To develop its P. falciparum sporozoite (SPZ) products, Sanaria has industrialized the production of P. falciparum-infected Anopheles stephensi mosquitoes, incorporating quantitative analyses of oocyst and P. falciparum SPZ infections as part of the manufacturing process.MethodsThese capabilities were exploited to develop a robust, reliable, consistent SMFA that was used to assess 188 serum samples from animals immunized with the candidate vaccine immunogen, Pfs25, targeting P. falciparum mosquito stages. Seventy-four independent SMFAs were performed. Infection intensity (number of oocysts/mosquito) and infection prevalence (percentage of mosquitoes infected with oocysts) were compared between mosquitoes fed cultured gametocytes plus normal human O(+) serum (negative control), anti-Pfs25 polyclonal antisera (MRA39 or MRA38, at a final dilution in the blood meal of 1:54 as positive control), and test sera from animals immunized with Pfs25 (at a final dilution in the blood meal of 1:9).ResultsSMFA negative controls consistently yielded high infection intensity (mean = 46.1 oocysts/midgut, range of positives 3.7-135.6) and infection prevalence (mean = 94.2%, range 71.4-100.0) and in positive controls, infection intensity was reduced by 81.6% (anti-Pfs25 MRA39) and 97.0% (anti-Pfs25 MRA38), and infection prevalence was reduced by 12.9 and 63.5%, respectively. A range of TBAs was detected among the 188 test samples assayed in duplicate. Consistent administration of infectious gametocytes to mosquitoes within and between assays was achieved, and the TBA of anti-Pfs25 control antibodies was highly reproducible.ConclusionsThese results demonstrate a robust capacity to perform the SMFA in a medium-to-high throughput format, suitable for assessing large numbers of experimental samples of candidate antibodies or drugs

Enterobacter-activated mosquito immune responses to Plasmodium involve activation of SRPN6 in Anopheles stephensi.

Successful development of Plasmodium in the mosquito is essential for the transmission of malaria. A major bottleneck in parasite numbers occurs during midgut invasion, partly as a consequence of the complex interactions between the endogenous microbiota and the mosquito immune response. We previously identified SRPN6 as an immune component which restricts Plasmodium berghei development in the mosquito. Here we demonstrate that SRPN6 is differentially activated by bacteria in Anopheles stephensi, but only when bacteria exposure occurs on the lumenal surface of the midgut epithelium. Our data indicate that AsSRPN6 is strongly induced following exposure to Enterobacter cloacae, a common component of the mosquito midgut microbiota. We conclude that AsSRPN6 is a vital component of the E. cloacae-mediated immune response that restricts Plasmodium development in the mosquito An. stephensi

<i>E. cloacae</i>-mediated inhibition of <i>P. falciparum</i> development is reversed by SRPN6 silencing.

<p>(A) Semi-quantitative RT-PCR analysis was used to determine the effects of gene silencing on <i>An. stephensi</i> midgut samples after feeding with a <i>P. falciparum</i> gametocyte/<i>E. cloacae</i> (10⁶/ml) mixture. SRPN6 mRNA abundance in midguts of dsSRPN6-injected mosquitoes was suppressed when compared with dsGFP controls. Ribosomal protein S7 (rpS7) served as a loading control. (B) <i>An. stephensi</i> mosquitoes were fed on a <i>P. falciparum</i> gametocyte culture (control) or were injected with dsGFP or dsSRPN6 and fed a <i>P. falciparum</i> gametocyte/<i>E. cloacae</i> (10⁶/ml) mixture. Midgut oocyst numbers were determined after 8 days by staining with mercurochrome. Data were pooled from three independent experiments and analyzed using Kruskal-Wallis analysis and a Dunn’s post-test to determine significance. Median oocyst numbers are depicted by the red line and the total numbers (n) of individual mosquitoes analyzed are denoted below each sample. The presence (+) or absence (−) of <i>Enterobacter</i> feeding or dsRNA treatment are shown below each sample. <i>P</i>-values are denoted by asterisks (** = <i>P</i><0.01; *** = <i>P</i><0.001). (C) Bar graphs showing the prevalence of infection among samples shows that SRPN6-silencing increase the percentage of mosquitoes containing at least one <i>P. falciparum</i> oocyst, although the results are not significant when analyzed by Chi-squared analysis.</p

<i>E. cloacae</i> inhibits <i>P. falciparum</i> development in <i>An. stephensi</i>.

<p>(A) Mosquitoes were fed on a <i>P. falciparum</i> gametocyte culture mixed either with medium alone (control) or with <i>E. cloacae</i> (+<i>Ec</i>; at a final concentration of 1×10⁶/ml). After 8 days, oocyst numbers were counted by mercurochrome staining of dissected midgut samples and the data were pooled from four independent experiments. Median oocyst numbers are depicted by the red line and the <i>P</i>-value was determined using a Mann–Whitney U test. The total numbers (n) of mosquitoes analyzed are denoted below each sample. The percentage of mosquitoes containing at least one <i>P. falciparum</i> oocyst (or prevalence of infection) is shown in (B). Samples were analyzed by Chi-squared analysis to determine significance. <i>P</i>-values are denoted by asterisks (* = <i>P</i><0.05; *** = <i>P</i><0.001).</p

SRPN6 expression following bacterial injection into the mosquito hemocoel.

<p>Approximately 2×10³ bacteria were injected into the hemocoel of adult female <i>An. stephensi.</i> SRPN6 expression was analyzed by Northern blot in the midgut (A) and carcass (all non-gut tissues) (B) 6 h post bacteria injection of into the hemocoel. Procedures and abbreviations are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062937#pone-0062937-g001" target="_blank">Figure 1A</a>. Similar results were obtained in three independent experiments. (C) As a control, expression of the anti-microbial peptide defensin was monitored by semi-quantitative RT-PCR in the carcass samples after bacteria injection. Procedures and abbreviations are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062937#pone-0062937-g001" target="_blank">Figure 1B</a>.</p

SRPN6 is differentially induced by bacteria in the mosquito midgut.

<p>(A) Bacteria (1×10⁶/ml of buffer; 2,000 bacteria assuming ingested volume of 2 µl) or the indicated component were fed to <i>An. stephensi</i> mosquitoes and their midguts were dissected 6 h later. Total RNA (3 µg) was analyzed by Northern blot using a ³²P-labeled SRPN6 cDNA probe (upper panel). The blot was then stripped and hybridized with mitochondrial rRNA probe as a loading control (lower panel). Samples are identified above each lane as follows. U: unfed control; B: buffer-fed; Lp: <i>E. coli</i> LPS (10 mg/ml); Ec: <i>Enterobacter cloacae</i>; St: <i>Salmonella typhimurium</i>; Sm: <i>Serratia marcescens</i>; Pa: <i>Pseudomonas aeruginosa</i>; Ml: <i>Micrococcus luteus</i>; Bs: <i>Bacillus subtilis</i>; Sa: <i>Staphylococcus aureus</i>; Pb: <i>P. berghei</i>-infected blood, analyzed 24 h after feeding (positive control). (B) Expression of gambicin, a mosquito anti-microbial peptide. Gambicin transcript abundance was analyzed by semi-quantitative RT-PCR (upper panel) using ribosomal protein S7 (rpS7) mRNA expression as a loading control (lower panel). RNA templates are the same as those used in panel (A).</p

Effect of <i>E. cloacae</i> on <i>P. falciparum</i> ookinete development in the mosquito.

<p><i>An. stephensi</i> females were fed <i>P. falciparum</i> gametocyte cultures mixed with medium alone (control) or with <i>E. cloacae</i> at the indicated concentrations. Individual midguts were dissected 20 h later and the number of ookinetes per gut was determined. Each value is the mean number of <i>P. falciparum</i> ookinetes (±SD) obtained from five midguts (n = 5) under each experimental condition. Each experiment was individually analyzed using a One-way ANOVA with a Dunnett’s post-test to determine the effects of <i>E. cloacae</i> on <i>P. falciparum</i> ookinete development. Asterisks denote significant differences with a <i>P</i> value of <0.05.</p

Activation of SRPN6 midgut expression by <i>E. cloacae</i>.

<p>(A) Time course of <i>An. stephensi</i> SRPN6 mRNA expression after feeding of <i>E. cloacae</i> (1×10⁶/ml; open bars) or <i>P. berghei</i> (green bars) as determined by qRT-PCR using ribosomal protein S7 (rpS7) for normalization. Values are reported in fold change relative to expression before feeding (0 h). (B) Immunolocalization of SRPN6 in midguts of mosquitoes fed with buffer (left panels) or <i>E. cloacae</i> (right panels). Guts were dissected 6 h after feeding, opened up into sheets, fixed and the protein detected with an anti-SRPN6 antibody. The inserts show higher magnifications of the areas within the squares. (C) Western blot analysis of SRPN6 protein expression after feeding with buffer alone (B) or at 6 and 24 h after <i>E. cloacae</i> ingestion, as indicated. Recombinant SRPN6 protein was used as a positive control (Rec). The blot was stripped and re-probed with an anti-actin antibody as a loading control (lower panel).</p

Regulation of Anti-<em>Plasmodium</em> Immunity by a <em>LITAF</em>-like Transcription Factor in the Malaria Vector <em>Anopheles gambiae</em>

<div><p>The mosquito is the obligate vector for malaria transmission. To complete its development within the mosquito, the malaria parasite <em>Plasmodium</em> must overcome the protective action of the mosquito innate immune system. Here we report on the involvement of the <em>Anopheles gambiae</em> orthologue of a conserved component of the vertebrate immune system, LPS-induced TNFα transcription factor (<em>LITAF</em>), and its role in mosquito anti-<em>Plasmodium</em> immunity. <em>An. gambiae LITAF</em>-like 3 (<em>LL3</em>) expression is up-regulated in response to midgut invasion by both rodent and human malaria parasites. Silencing of <em>LL3</em> expression greatly increases parasite survival, indicating that <em>LL3</em> is part of an anti-<em>Plasmodium</em> defense mechanism. Electrophoretic mobility shift assays identified specific LL3 DNA-binding motifs within the promoter of <em>SRPN6</em>, a gene that also mediates mosquito defense against <em>Plasmodium</em>. Further experiments indicated that these motifs play a direct role in LL3 regulation of <em>SRPN6</em> expression. We conclude that <em>LL3</em> is a transcription factor capable of modulating <em>SRPN6</em> expression as part of the mosquito anti-<em>Plasmodium</em> immune response.</p> </div

Immunofluorescence localization of LL3 in the mosquito midgut.

<p>To determine the localization of LL3 following <i>P. berghei</i> infection, midgut sheets were prepared 20 h PBM and visualized using a peptide-derived LL3 antibody. (A) Expression of LL3 (red) was detected in close proximity to invading ookinetes. Images are displayed as LL3 alone (left panel) or as a merged image (right panel) with ookinetes detected by an α-aldolase antibody (green) and DAPI staining (blue) to denote nuclei. (B) dsRNA-mediated silencing of LL3 (dsLL3) correlates with a dramatic reduction in the LL3 signal in comparison with dsGFP controls. Colors of the signals are as indicated on top of each panel. (C) Localization of LL3 following pervanadate treatment. LL3 protein staining was measured in control midgut sheets (−PV) or in midguts following treatment with pervanadate (+PV). Images are displayed as LL3 alone or as a merged image with DAPI staining as indicated on the top of each panel. All images are representative of multiple biological replicates. Scale bars denote 20 microns in all images.</p