Western blot analysis of CSP expression and processing in transgenic midgut sporozoites 18 days post-infection.

<p>CSP expression was revealed by incubation with either a monoclonal antibody directed against the PbCSP repeat region (PbCS-RR) or a serum directed against the PgCSP N-terminal region (PgCS-Nt). As a control, midgut lysates from uninfected mosquitoes were also analysed. Antibody against the PbCSP repeat region reveals a higher molecular weight precursor polypeptide and a lower molecular weight processed polypeptide. A ladder of degradation products is also visible. Antibody against the PgCSP N-terminal region reveals only the higher molecular weight protein.</p

Generation and southern blot analysis of transgenic PgCS/Pb^RR parasite lines.

<p>(<b>A</b>) Schematic representation of (i) the PgCS/Pb^RR targeting construct, (ii) the wt <i>PbCS</i> locus and (iii) the targeted locus after recombination between the <i>PbCS</i> 5′UTR and 3′UTR sequences. The vertically dashed box indicates the 1.13 kb 5′UTR sequence used in the construct and the horizontally dashed boxes indicate the 0.3 kb and 0.85 kb 3′UTR sequences between which the TgDHFR-TS selectable marker cassette (light grey) was inserted in the construct. The PgCS/Pb^RR chimeric gene contains the <i>PgCS</i> N- and C-terminal regions (dark grey boxes) and the <i>PbCS</i> repeat region (white box) flanked by the <i>Spe</i>I (<i>S</i>) and <i>Xho</i>I (<i>X</i>) sites. In the wt PbCS locus the white box indicates the full <i>PbCS</i> gene. Thick black lines indicate the probes used in southern blots. E: <i>EcoR</i>V site. (<b>B</b>) Southern blot of <i>EcoR</i>V/<i>Spe</i>I (E/S) and <i>EcoR</i>V/<i>Xho</i>I (E/X) digested genomic DNA from Pbwt parasites and transgenic PgCS/Pb^RR parasites (clones 4 and 5), hybridised with the <i>PbCS</i> 3′UTR probe. Two bands of 4.0 and 1.5 kb were present in Pbwt DNA in both digestions. E/S digested DNA from the PgCS/Pb^RR clones revealed two bands of 1.1 and 2.0 kb, while E/X digested DNA revealed two bands of 0.6 and 2.0 kb, demonstrating the replacement of the endogenous <i>PbCS</i> gene with the targeted construct. (<b>C</b>) The same membrane was then hybridised with the <i>PgCS</i> N-terminal probe (PgCS-Nt), encompassing the entire N-terminal region of <i>PgCS</i>. No band was present in Pbwt DNA. However, a band of 3.1 kb or 3.6 kb was present in the E/S or E/X digested transgenic DNA, respectively.</p

Sporozoite CSP expression.

<p>Confocal immunofluorescence microphotographs of transgenic midgut sporozoites incubated at room temperature and developed with either a monoclonal antibody directed against the PbCSP repeat region (Pb-RR), a serum directed against the PgCSP N-terminal region (Pg-Nt), or a serum directed against the PgCSP repeat region (Pg-RR). Antibodies against the PbCSP and PgCSP repeat regions showed surface expression of the CSPs, whereas antibody against the PgCSP N-terminal region revealed mainly an intracellular pattern of expression.</p

Transgenic parasite development in the <i>Anopheles stephensi</i> mosquito vector.

a<p>Significant difference compared to Pbwt and PbCS^DHFR transgenic parasites; p<0.01.</p>b<p>Significant difference compared to PgCS^SX; p<0.03.</p><p><i>A. stephensi</i> mosquitoes were fed on mice infected with one of the transgenic clones or Pbwt parasites. Values represent the mean ± S.D. of at least three independent experiments, each with a minimum of 50 mosquitoes.</p

Infectivity of midgut sporozoites for the vertebrate host.

a<p>Pre-patent period is the number of days between injection and first appearance of the parasites in the peripheral blood.</p><p>C57BL/6 mice were injected intravenously with Pbwt or transgenic midgut sporozoites collected on day 21 p.i.. The number of mice that became infected and the pre-patent period were both recorded.</p

Schematic representation and alignment of wild type and transgenic <i>P.berghei</i> circumsporozoite proteins.

<p>(A) Schematic representation of the CS proteins present in each of the four transgenic <i>P. berghei</i> parasite lines generated. The corresponding names of the transgenic parasite lines are shown on the right. PbCS^DHFR parasites carry the wildtype PbCS coding sequence (white boxes, RI and RII indicated with light and dark green respectively) and, similar to all transgenic lines, contain the <i>T. gondii</i> DHFR drug selectable marker inserted in the CS locus. PgCS^SX parasites carry the full PgCS coding sequence (grey boxes, RI and RII indicated with light and dark blue respectively), but with the <i>Spe</i>I (S) and <i>Xho</i>I (X) restriction endonuclease sites inserted on either side of the repeat region. PgCS/Pb^RR parasites contain the PgCS N-terminal and C-terminal regions (grey boxes) and the PbCS repeat region (white box). PbCS/Pg^CT parasites carry the PbCS N-terminal and repeat regions (white boxes) and the PgCS C-terminal region (grey box). (B) Alignment of the wild type PbCS and transgenic PgCS^SX and PgCS/Pb^RR amino acid sequences. Shaded boxes represent areas of amino acid identity. Regions I and II (black lines), the repeat region (blue line) and the <i>Spe</i>I and <i>Xho</i>I restriction sites (red lines) are labelled. The <i>Spe</i>I and <i>Xho</i>I sites were introduced into the PgCS sequence to mediate exchange of the PgCS repeat region with the PbCS repeat region.</p

Effect of halofuginone on vaginal infection.

<p>Mice, under pseudo-estrus conditions, were twice infected with 10⁷<i>Candida albicans</i> in vagina. Two days before and every two days after infection, mice were injected intraperitoneally with 5 µg/100 µl or 10 µg/100 µl of halofuginone solution or diluent of halofuginone and, in selected experiments, were treated intravaginally with 10 pg of mouse rIL-17. (A) Evaluation of IL-17 concentration by ELISA in supernatants of vaginal fluids obtained at different days after vaginal <i>Candida</i> infection and halofuginone treatment. Results are expressed as mean±SD (n = 9 mice, 3 mice for each of three separate experiments). The statistical analysis was performed using Mann-Whitney U test. * <i>p</i><0.05, ** <i>p</i><0.01 (infected halofuginone treated mice vs infected diluent treated mice). At day 4, 14 and 25 after infection, mice were treated intravaginally with 10 µg of coelenterazine and imaged in the IVIS-200™ imaging system under anesthesia using 2.5% isoflurane and the vaginal lumen was washed with 150 µl of saline. (B) In vivo imaging of mice vaginally infected with <i>Candida albicans</i> cells (gLUC) and treated with halofuginone or diluent. Images are representative of 5 out of 10 mice in two different experiments. (C) Dot plot of total photon emission from the infected regions and dot plot of CFU in vaginal washes of mice (n = 10) treated with halofuginone or diluent. The statistical analysis was performed using non-parametric Mann-Whitney U test. The median is indicated by a straight line. Data are representative of one out of two independent experiments with similar results. * <i>p</i><0.05, ** <i>p</i><0.01 (infected halofuginone treated mice vs infected diluent treated mice).</p

IL-17 and IL-23 concentration in murine vaginal washes of mice infected with <i>Candida albicans</i>.

<p>Evaluation of IL-17 (A–C) and IL-23 (D) concentration by ELISA test on supernatants of vaginal fluids obtained at different times after vaginal infection with different doses of <i>Candida albicans</i> gLUC59 (A–B) or CA1399 (D). Results are expressed as mean±SD (n = 12 mice, 4 mice for each of three separate experiments). * <i>p</i><0.05, (infected mice vs non infected mice).</p

Effect of <i>Candida albicans</i> infection on draining lumbar lymph nodes.

<p>Lymph nodes, at different times after <i>Candida</i> infection, were aseptically recovered and mechanically homogenized. Cells were cultivated untreated or in presence of heat inactivated <i>C. albicans</i> for 72 hours. In the supernatant fluids of lymph node cell culture IL-17 (A) and IL-23 (B) were analyzed by ELISA. Results are expressed as mean±SD (n = 16 mice, 4 mice for each of four separate experiments). The statistical analysis was performed using Mann-Whitney U test. * <i>p</i><0.05, (Lymphocytes from infected mice vs Lymphocytes from non infected mice). # <i>p</i><0.05, ## <i>p</i><0.01, (Lymphocytes re-stimulated from infected mice vs Lymphocytes re-stimulated from non infected mice).</p

Model of murine vaginal infection and monitoring.

<p>(A) Timeline of vaginal infection model. CD1 mice are resistant to <i>Candida</i> vaginal infection unless they are treated with estradiol valerate. CD1 mice were treated subcutaneously with estradiol valerate and infected for two consecutive days with 10 µl of 10⁹/ml suspension of <i>Candida albicans</i> cells (gLUC) into vaginal lumen. Two days before and every two days after challenge mice were treated intraperitoneally with halofuginone or diluent of halofuginone and, in selected experiments, intravaginally with 10 pg of recombinant mouse IL-17. After 4, 8, 12, 14, 18, 20, 25, 30 days post infection, mice were treated intravaginally with 10 µg of coelenterazine and imaged in the IVIS-200™ imaging system under anaesthesia using 2.5% isoflurane and the vaginal lumen was washed with 150 µl of saline using mechanical pipette. The fungal burden of vaginal fluids was evaluated by colony forming units (CFU) assay. (B) In vivo imaging of mice vaginally infected with <i>Candida albicans</i> cells (gLUC). Images are representative of 5 out of 10 mice for each experiment. C) Dot plots of total photon emission from the infected vaginal regions and corresponding CFU in vaginal washes of infected mice (n = 10). The statistical analysis was performed by non-parametric Mann-Whitney U test. The median is indicated by a straight line. Data are representative of one of two independent experiments with similar results. D) The correlation between the Total Photons emitted and CFU count in the vaginal wash was assessed using the Pearson's correlation statistics, and the correlation coefficients are shown for each time point. * <i>p</i><0.05, ** <i>p</i><0.01, (Log Photons Total Flux or Log CFU/ml of mice infected after 8, 12,14,18,20,25,30 days vs Log Photons Total Flux or Log CFU/ml of mice infected after 4 days).</p