Journal: PLOS Pathogens

Article Title: Vibrio cholerae cytolysin induces pro-inflammatory and death signals through novel TLR assembly

doi: 10.1371/journal.ppat.1013033

Figure Lengend Snippet: (A-B) Immunoblot analyses to probe TLR2 co-immunoprecipitation with VCC in BMDCs, treated with VCC monomer (A) or pre-formed VCC oligomer (B). Cells were treated with VCC monomer (10 ng/ml; A) or pre-formed VCC oligomer (1 μg/ml; B) for 30 minutes. Cell lysates were subjected to immunoprecipitation (IP) with anti-VCC antibody, and immunoprecipitated fractions were subsequently probed for TLR2 and VCC by immunoblotting (IB) using anti-TLR2 and anti-VCC antibodies, respectively. β-actin was probed as the pre-IP protein loading control. Buffer and liposome-treated cells served as the negative controls for the experiments with VCC monomer and oligomer, respectively. Data shown here are the representatives of three independent experiments. (C-D) VCC did not co-immunoprecipitate with TLR2 in BMDCs, treated with VCC monomer (C) or pre-formed VCC oligomer (D). BMDCs were treated with VCC monomer (10 ng/ml; C) or pre-formed oligomer (1 μg/ml; D) for 30 minutes. Cell lysates were subjected to immunoprecipitation (IP) with anti-TLR2 antibody, and immunoprecipitated fractions were probed for TLR2 and VCC by immunoblotting (IB) using anti-TLR2 and anti-VCC antibodies, respectively. TLR2 was probed as the protein loading control. Buffer and liposome-treated cells served as negative controls for the experiments with VCC monomer and oligomer, respectively. Immunoblots shown here are the representatives of three independent experiments. (E) No significant decrease in the VCC-induced IL-6 production in BMDCs from TLR2 -/- mice, as compared to that in BMDCs from the wild type (WT) mice. BMDCs from wild type (WT) and TLR2 -/- mice were stimulated with VCC monomer (10 ng/ml) or pre-formed VCC oligomer (1 μg/ml) for 24 hours. Following the treatments, IL-6 production was monitored by ELISA. Cells treated with buffer and liposomes served as the negative controls for the experiments with VCC monomer and oligomer, respectively. Data shown here are the averages ± SDs from three to four independent experiments. ns, non-significant; p > 0.05; one-way ANOVA with Tukey’s multiple comparison test. (F-G) Immunoblot analyses showing co-immunoprecipitation of TLR1 with VCC in BMDCs, treated with VCC monomer (F) or pre-formed VCC oligomer (G). Cells were treated with VCC monomer (10 ng/ml; F) or pre-formed VCC oligomer (1 μg/ml; G) for 30 minutes. Cell lysates were subjected to immunoprecipitation (IP) with anti-VCC antibody, and immunoprecipitated fractions were subsequently probed for TLR1 and VCC by immunoblotting (IB) using anti-TLR1 and anti-VCC antibodies, respectively. β-actin was probed as the pre-IP protein loading control. Buffer and liposome-treated cells served as the negative controls for the experiments with VCC monomer and oligomer, respectively. Data shown here are the representatives of three independent experiments. (H-I) VCC co-immunoprecipitates with TLR1 in BMDCs, treated with VCC monomer (H) or pre-formed VCC oligomer (I). BMDCs were treated with VCC monomer (10 ng/ml) or pre-formed VCC oligomer (1 μg/ml) for 30 minutes. Cell lysates were subjected to immunoprecipitation (IP) with anti-TLR1 antibody, and immunoprecipitated fractions were probed for TLR1 and VCC by immunoblotting (IB) using anti-TLR1 and anti-VCC antibodies, respectively. TLR1 was probed as the protein loading control. Buffer and liposome-treated cells served as negative controls for the experiments with VCC monomer and oligomer, respectively. Immunoblots shown here are the representatives of three independent experiments. (J) siRNA-mediated knock-down of TLR1 leads to a decrease in IL-6 production in BMDCs, upon treatment of the cells with pre-formed VCC oligomer. Cells were transfected with 75 nM siRNA against TLR1 or non-target control (scramble). Knock-down of TLR1 was confirmed using RT-qPCR after 48 hours of transfection (left panel; showing the fold change in the Tlr1 level over the untreated untransfected control). After 48 hours, cells were treated with 1 μg/ml pre-formed oligomer, and incubated for 12 hours. Subsequently, the cell supernatant was analysed for IL-6 production by ELISA (right panel). Scramble-transfected cells upon VCC oligomer treatment served as the siRNA control. Liposome-treated cells were also analysed as control. Data shown here are the averages ± SDs from three independent experiments. ns, non-significant; *, p < 0.05; Student’s unpaired t-test (for the TLR1 expression data) and one-way ANOVA with Tukey’s multiple comparison test (for the IL-6 production data). (K) Significant decrease in the IL-6 production in the VCC-treated BMDCs from TLR1 -/- mice, as compared to those from the wild type (WT) mice. BMDCs from wild type (WT) mice and TLR1 -/- mice were treated with VCC monomer (10 ng/ml) or pre-formed VCC oligomer (1 μg/ml) for 24 hours. After the incubation, IL-6 production was monitored using ELISA. Buffer and liposome-treated cells served as the negative controls for the experiments with VCC monomer and VCC oligomer, respectively. Data shown here are the averages ± SDs from three independent experiments. *, p < 0.05; **, p < 0.01; one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: For probing TLR1 surface expression, cells were incubated with anti-TLR1 antibody (1:200, volume/volume) (Santa Cruz Biotechnology) for 40-45 minutes, and then with Alexa Flour 488-conjugated secondary antibody (1:400, volume/volume) (Thermo Fisher Scientific) for 30 minutes on ice.

Techniques: Western Blot, Immunoprecipitation, Control, Enzyme-linked Immunosorbent Assay, Liposomes, Comparison, Knockdown, Transfection, Quantitative RT-PCR, Incubation, Expressing

Journal: PLOS Pathogens

Article Title: Vibrio cholerae cytolysin induces pro-inflammatory and death signals through novel TLR assembly

doi: 10.1371/journal.ppat.1013033

Figure Lengend Snippet: (A-B) Immunoblot analyses showing association of VCC with TLR4 along with MyD88 and TLR1 in BMDCs, treated with VCC monomer and pre-formed VCC oligomer. BMDCs were treated with 10 ng/ml VCC monomer (A) and 1 μg/ml pre-formed VCC oligomer (B) for 30 minutes. The whole-cell lysates from the treated cells were subjected to immunoprecipitation (IP) with anti-VCC antibody, and the immunoprecipitated fractions were probed by immunoblotting (IB) with anti-VCC, anti-TLR4, anti-TLR1, and anti-MyD88 antibodies. β-actin was probed as the pre-IP protein loading control. Buffer and liposome-treated cells served as controls for the experiments with VCC monomer and oligomer, respectively. Immunoblots shown are the representatives of three independent experiments. (C-D) VCC, MyD88, and TLR1 co-immunoprecipitated with TLR4 in the BMDCs, upon treatment with VCC monomer and pre-formed VCC oligomer. BMDCs were treated with 10 ng/ml VCC monomer (C) or 1 μg/ml pre-formed VCC oligomer (D) for 30 minutes. The whole-cell lysates from the treated cells were subjected to immunoprecipitation (IP) with anti-TLR4 antibody, and the immunoprecipitated fractions were probed by immunoblotting (IB) using anti-VCC, anti-TLR4, anti-TLR1, and anti-MyD88 antibodies. TLR4 served as the protein loading control. Buffer and liposome-treated cells served as the controls for the experiments with VCC monomer and oligomer, respectively. Immunoblots shown here are the representatives of three independent experiments. (E-F) Significant decrease in the VCC-mediated IL-6 production in BMDCs, upon pre-neutralization of TLR4 with anti-TLR4 antibody. BMDCs were pre-treated with anti-TLR4 neutralizing antibody for 2 hours. Following incubation, cells were treated with 10 ng/ml VCC monomer (E) or 1 μg/ml pre-formed VCC oligomer (F), and incubated for 24 hours. After the treatments, IL-6 levels in the extra-cellular supernatants were monitored by ELISA. Cells pre-treated with isotype antibody served as the control for the neutralizing antibody. Buffer and liposome-treated cells served as the negative controls for the experiments with VCC monomer and oligomer, respectively. Data shown here are the averages ± SDs from three independent experiments. ns, non-significant; *, p < 0.05; ***, p < 0.001; one-way ANOVA with Tukey’s multiple comparison test. (G-H) Significant decrease in the VCC-mediated IL-6 production in the BMDCs from the C3H/HeJ mice with abrogated TLR4 signalling, compared to that in the BMDCs from the wild type (WT) mice. BMDCs from the wild type (WT) and C3H/HeJ mice were treated with 10 ng/ml VCC monomer (G) or 1 μg/ml pre-formed VCC oligomer (H) for 24 hours. Following incubation, supernatants were collected and IL-6 production was estimated by ELISA. Buffer and liposome-treated cells served as the negative controls for the experiments with VCC monomer and oligomer, respectively. Data shown here are the averages ± SDs from three independent experiments. **, p < 0.01; one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: For probing TLR1 surface expression, cells were incubated with anti-TLR1 antibody (1:200, volume/volume) (Santa Cruz Biotechnology) for 40-45 minutes, and then with Alexa Flour 488-conjugated secondary antibody (1:400, volume/volume) (Thermo Fisher Scientific) for 30 minutes on ice.

Techniques: Western Blot, Immunoprecipitation, Control, Neutralization, Incubation, Enzyme-linked Immunosorbent Assay, Comparison

Journal: PLOS Pathogens

Article Title: Vibrio cholerae cytolysin induces pro-inflammatory and death signals through novel TLR assembly

doi: 10.1371/journal.ppat.1013033

Figure Lengend Snippet: (A-B) HEK293 cells were transfected with NF-κB Luciferase reporter plasmid, pRL plasmid, along with pcDNA3.1(+)-TLR1, pcDNA3.1(+)-TLR2, pcDNA3.1(+)-TLR4, pcDNA3.1(+)-TLR6, and empty pcDNA3.1(+), in different combinations. After 24 hours, cells were stimulated with 100 ng/ml VCC monomer (A) or 1 μg/ml pre-formed VCC oligomer (B) for additional 12 hours. Empty pcDNA3.1(+)-transfected, VCC-treated cells served as the controls. The NF-κB activation was measured using the Luciferase Reporter Assay. The reporter activity (Relative Luminescence Unit (R.L.U)) was presented in terms of fold change over that in the pcDNA3.1(+)-transfected, VCC-treated cells. Data shown here are the averages ± SDs from three independent experiments. **, p < 0.01; ***, p < 0.001; one-way ANOVA with Dunnett’s multiple comparison test. (C-D) HEK293 cells were transfected with NF-κB Luciferase reporter plasmid, pRL (renilla) plasmid, along with pcDNA3.1(+)-MD-2, pcDNA3.1(+)-CD14, pcDNA3.1(+)-TLR4, pcDNA3.1(+)-TLR1, and empty pcDNA3.1(+), in different combinations. After 24 hours, cells were treated with 100 ng/ml VCC monomer (C), 1 μg/ml pre-formed VCC oligomer (D), or 200 ng/ml LPS (C-D), and the NF-κB reporter activity was measured after 12 hours of the treatments, as described above (in A-B). Empty pcDNA3.1(+)-transfected, VCC-treated cells served as the control. Data shown here are the averages ± SDs from three independent experiments. ***, p < 0.001; one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: For probing TLR1 surface expression, cells were incubated with anti-TLR1 antibody (1:200, volume/volume) (Santa Cruz Biotechnology) for 40-45 minutes, and then with Alexa Flour 488-conjugated secondary antibody (1:400, volume/volume) (Thermo Fisher Scientific) for 30 minutes on ice.

Techniques: Transfection, Luciferase, Plasmid Preparation, Activation Assay, Reporter Assay, Activity Assay, Comparison, Control

Journal: PLOS Pathogens

Article Title: Vibrio cholerae cytolysin induces pro-inflammatory and death signals through novel TLR assembly

doi: 10.1371/journal.ppat.1013033

Figure Lengend Snippet: Significant decrease in the VCC-mediated IL-6 production in BMDMs from TLR2 -/- (A-B), C3H/HeJ (A-B), and TLR1 -/- (C-D) mice, as compared to that in BMDMs from the wild type (WT) mice. BMDMs from wild type (WT), TLR2 -/- , C3H/HeJ, and TLR1 -/- mice were treated with 10 ng/ml VCC monomer (A, C) and 1 μg/ml VCC oligomer (B, D). After 24 hours of treatment, the supernatants were analysed for the IL-6 production using ELISA. Buffer and liposome-treated cells served as negative controls for the experiment with VCC monomer and oligomer, respectively. Data shown here are the averages ± SDs from three to four independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: For probing TLR1 surface expression, cells were incubated with anti-TLR1 antibody (1:200, volume/volume) (Santa Cruz Biotechnology) for 40-45 minutes, and then with Alexa Flour 488-conjugated secondary antibody (1:400, volume/volume) (Thermo Fisher Scientific) for 30 minutes on ice.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: PLOS Pathogens

Article Title: Vibrio cholerae cytolysin induces pro-inflammatory and death signals through novel TLR assembly

doi: 10.1371/journal.ppat.1013033

Figure Lengend Snippet: (A) Decrease in serum IL-6 levels in TLR1 -/- , TLR2 -/- , C3H/HeJ and MyD88 -/- mice, in comparison to that in the wild type (WT) mice upon intra-peritoneal administration of VCC. WT, TLR1 -/- , TLR2 -/- , C3H/HeJ and MyD88 -/- mice were injected intra-peritoneally with 5 μg VCC (in 100 μl PBS), and blood was collected after 4 hours. Serum was obtained, and IL-6 level in the serum was estimated by ELISA. WT and the transgenic mice injected with equal volume of PBS served as the negative controls. Data shown here are the averages ± SDs from three independent experiments. ***, p < 0.001; one-way ANOVA with Tukey’s multiple comparison test. (B) Left panel shows the decrease in serum IL-6 levels in mice infected with VCC-deleted (Δ hlyA ) V. cholerae variant, in comparison to those in mice infected with the wild type (WT) V. cholerae . Right panel shows the colonization efficacies of WT and Δ hlyA V. cholerae . Mice were infected orally with the bacteria (5x10 9 cells). After 4 hours of infection, serum was obtained from the blood, and IL-6 level in the serum was estimated by ELISA (left panel). For the colonization assay, mice stools were collected for 10 consecutive days after the infection, weighed, and plated on the TCBS agar. Line graph represents the Log10 cfu/gram of the stool for 10 days (right panel). Data shown here are the averages ± SDs from three (for the serum IL-6 measurements) to four (for the colonization assay) independent experiments. ***, p < 0.001; Student’s unpaired t-test. (C) Decrease in the serum IL-6 levels in TLR1 -/- , C3H/HeJ and MyD88 -/- mice, in comparison to that in the wild type (WT) mice upon oral infection with wild type (WT) V. cholerae . Wild type (WT), TLR1 -/- , C3H/HeJ and MyD88 -/- mice were orally infected with V. cholerae , and blood was collected after 4 hours. Serum was obtained, and IL-6 level in the serum was estimated by ELISA. Data shown here are the averages ± SDs from three or more independent experiments. **, p < 0.01; ***, p < 0.001; one-way ANOVA with Dunnett’s multiple comparison test. (D) Increased survival of TLR1 -/- , C3H/HeJ and MyD88 -/- mice, as compared to that of the WT mice, upon VCC intoxication. WT, TLR1 -/- , TLR2 -/- , C3H/HeJ and MyD88 -/- mice were injected intra-peritoneally with 5 μg VCC (in 100 μl PBS), and the survival of the animals was monitored. Kaplan-Meier plot of cumulative mortality is presented to compare the survival rate. WT and transgenic mice injected with equal volume of PBS only served as the control. For the VCC-treated mice in each group, n=7 (1 mouse/experiment was taken in each group, and the experiment was performed 7 times). For the negative control, 1 mouse/experiment was also taken in each group that was injected with PBS (n=7). However, for the simplified presentation purpose, the survival time-point for one PBS-treated control mouse from each group is shown in the graph.

Article Snippet: For probing TLR1 surface expression, cells were incubated with anti-TLR1 antibody (1:200, volume/volume) (Santa Cruz Biotechnology) for 40-45 minutes, and then with Alexa Flour 488-conjugated secondary antibody (1:400, volume/volume) (Thermo Fisher Scientific) for 30 minutes on ice.

Techniques: Comparison, Injection, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Infection, Variant Assay, Bacteria, Control, Negative Control

Journal: PLOS Pathogens

Article Title: Vibrio cholerae cytolysin induces pro-inflammatory and death signals through novel TLR assembly

doi: 10.1371/journal.ppat.1013033

Figure Lengend Snippet: (A-B) LDH-release assay of cytotoxicity showing the extent of VCC-induced cell death in BMDCs (A) and BMDMs (B) from wild type (WT), TLR1 -/- , TLR2 -/- , C3H/HeJ, and MyD88 -/- mice. BMDCs/BMDMs from WT and transgenic mice were treated with 10 ng/ml VCC monomer for 12 hours. Following treatments, supernatants were collected, and %cytotoxicity was measured by LDH-release assay. Buffer-treated cells served as the negative controls. Data shown here are the averages ± SDs of %cytotoxicity from three independent treatments. ns, non-significant; **, p < 0.01; ***, p < 0.001; one-way ANOVA with Tukey’s multiple comparison test. (C-D) Confocal microscopy images showing the extent of cell death in BMDCs (C) and BMDMs (D) from wild type (WT), TLR1 -/- , TLR2 -/- and MyD88 -/- mice, upon co-culturing in the presence of V. cholerae (lower panels in C and D). BMDCs/BMDMs were co-cultured with V. cholerae for 7 hours. Uninfected cells served as the controls (upper panels in C and D). Cells were stained with CellTrace (green), and cell death was visualized with propidium iodide (PI) staining (red). A total of 15 to 20 images, from three (for BMDCs) to four (for BMDMs) independent experiments, were taken for each of the uninfected and infected cells from the WT, TLR1 -/- , TLR2 -/- and MyD88 -/- mice, and the representative images are shown here. Bar graphs (in the right) show the percentage of cell death in the V. cholerae -infected BMDCs (in C) and BMDMs (in D) from the WT, TLR1 -/- , TLR2 -/- and MyD88 -/- mice. Data shown here are the averages ± SDs. ns, non-significant; *, p<0.05; ***, p < 0.001; one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: For probing TLR1 surface expression, cells were incubated with anti-TLR1 antibody (1:200, volume/volume) (Santa Cruz Biotechnology) for 40-45 minutes, and then with Alexa Flour 488-conjugated secondary antibody (1:400, volume/volume) (Thermo Fisher Scientific) for 30 minutes on ice.

Techniques: Lactate Dehydrogenase Assay, Transgenic Assay, Comparison, Confocal Microscopy, Cell Culture, Staining, Infection

Journal: PLOS Pathogens

Article Title: Vibrio cholerae cytolysin induces pro-inflammatory and death signals through novel TLR assembly

doi: 10.1371/journal.ppat.1013033

Figure Lengend Snippet: (A) VCC induces pro-inflammatory responses during V. cholerae infection of neutrophils. Neutrophils isolated from bone marrow were infected with either wild type (WT) or VCC-deleted (Δ hlyA ) variants of V. cholerae at the multiplicity of infection (MOI) of 10 for 4 hours. Supernatants were analysed for the production of IL-6 (left) and TNF-α (right) using ELISA. Data shown here are the averages ± SDs from three to five independent experiments. **, p < 0.01; Student’s unpaired t-test. (B) Increase in the IL-6 production by the neutrophils over time upon VCC treatment. Neutrophils isolated from the bone marrow were treated with 20 ng/ml VCC monomer for 2, 4, 8 and 12 hours. Following treatments, the supernatants were analysed for the IL-6 production. Buffer-treated cells served as the negative control. Data shown here are the averages ± SDs from the four independent experiments. ns, non-significant; **, p < 0.01; one-way ANOVA with Dunnett’s multiple comparison test. (C) Significant decrease in the VCC-mediated IL-6 production in the neutrophils from the TLR1 -/- , C3H/HeJ, and MyD88 -/- mice, as compared to those in neutrophils from the wild type (WT) mice. Neutrophils from the wild type (WT), TLR1 -/- , C3H/HeJ, and MyD88 -/- mice were treated with 20 ng/ml VCC monomer. After 12 hours of treatment, the supernatants were analysed for the IL-6 production using ELISA. Buffer-treated cells served as the negative controls. Data shown here are the averages ± SDs from four independent experiments. ***, p < 0.001; one-way ANOVA with Tukey’s multiple comparison test. (D) VCC-induced pro-inflammatory responses in neutrophils during the V. cholerae infection are TLR1, TLR4 and MyD88-dependent. Neutrophils isolated from the bone marrow of the wild type (WT), TLR1 -/- , C3H/HeJ, and MyD88 -/- mice were infected with wild type (WT) V. cholerae at an MOI of 10 for 4 hours. Supernatants were analysed for the production of TNF-α using ELISA. Data shown here are the averages ± SDs from four independent experiments. ns, non-significant; **, p < 0.01; ***, p < 0.001; one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: For probing TLR1 surface expression, cells were incubated with anti-TLR1 antibody (1:200, volume/volume) (Santa Cruz Biotechnology) for 40-45 minutes, and then with Alexa Flour 488-conjugated secondary antibody (1:400, volume/volume) (Thermo Fisher Scientific) for 30 minutes on ice.

Techniques: Infection, Isolation, Enzyme-linked Immunosorbent Assay, Negative Control, Comparison

Journal: Life Science Alliance

Article Title: Full-length MSP1 is a major target of protective immunity after controlled human malaria infection

doi: 10.26508/lsa.202301910

Figure Lengend Snippet: Spearman’s correlation of anti-MSP1 FL and anti-merozoite IgG, IgM, and IgG subclass 1–4 antibodies in CHMI volunteers (n = 142). Each data point represents antibody levels for one sample in duplicates.

Article Snippet: The Plasmid containing the codon-optimized sequence of full-length MSP1 (3D7) was received from the plasmid repository Addgene (#47709; Plasmid) which has been optimized for expression in mammalian cells ( ; ).

Techniques:

Journal: Life Science Alliance

Article Title: Full-length MSP1 is a major target of protective immunity after controlled human malaria infection

doi: 10.26508/lsa.202301910

Figure Lengend Snippet: (A) The prevalence of IgG antibodies in treated (T, n = 56) and non-treated volunteers (NT, n = 86). (B) IgG antibody levels against MSP1 subunits were compared between treated (T, n = 56) and non-treated volunteers (NT, n = 86). (C) Spearman’s correlation of anti-MSP1 FL (3D7) and MSP1 FL -F (K1) antibody levels (OD) for CHMI volunteers (n = 142). (D) IgG antibody levels against MSP1 FL -F were compared between treated (T, n = 56) and non-treated volunteers (NT, n = 86). Each data point represents antibody levels for one sample in duplicates measured in one experiment. The seropositivity cut-off value was calculated as the optical density (OD) of malaria-naïve plasma samples (n = 5) plus three standard deviations indicated as the dotted line. Error bars represent the median plus 95% confidence intervals. Statistical differences between treatment outcomes were calculated using the Mann-Whitney test.

Article Snippet: The Plasmid containing the codon-optimized sequence of full-length MSP1 (3D7) was received from the plasmid repository Addgene (#47709; Plasmid) which has been optimized for expression in mammalian cells ( ; ).

Techniques: Clinical Proteomics, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: Full-length MSP1 is a major target of protective immunity after controlled human malaria infection

doi: 10.26508/lsa.202301910

Figure Lengend Snippet: Fluorescence intensity landscapes across MSP1 FL shown for individuals of the four subgroups based on parasite growth patterns: treated febrile (n = 10), treated non-febrile (n = 10), non-treated PCR positive (n = 10), and non-treated PCR negative (n = 10). Every line represents a sample. Relevant epitopes have been highlighted on top. A graphical representation of the primary structure of MSP1 FL is shown below the fluorescence intensity landscapes.

Article Snippet: The Plasmid containing the codon-optimized sequence of full-length MSP1 (3D7) was received from the plasmid repository Addgene (#47709; Plasmid) which has been optimized for expression in mammalian cells ( ; ).

Techniques: Fluorescence

Journal: Life Science Alliance

Article Title: Full-length MSP1 is a major target of protective immunity after controlled human malaria infection

doi: 10.26508/lsa.202301910

Figure Lengend Snippet: (A) The prevalence of MSP1 FL -specific Fc-mediated functional activities in treated (T, n = 56) and non-treated volunteers (NT, n = 86). (B) The prevalence of MSP1 FL -specific Fc-mediated functional activities compared between the subgroups based on parasite growth patterns, treated febrile (F, n = 26), treated non-febrile (NF, n = 30), untreated PCR positive (PCR+, n = 53), and untreated PCR negative (PCR−, n = 33). (C) Levels of Fc-mediated effector functions of anti-MSP1 FL antibodies were compared between treated and non-treated volunteers. (D) Levels of Fc-mediated effector functions of anti-MSP1 FL antibodies were compared between the different subgroups, based on parasite growth densities: treated febrile, treated non-febrile, non-treated PCR positive, and non-treated PCR negative. Each data point represents antibody levels for one sample in duplicates measured in one experiment. The seropositivity cut-off value was calculated as the activity level of malaria-naïve plasma samples (n = 5) plus three standard deviations indicated as the dotted line. Error bars represent the median plus 95% confidence intervals. Statistical differences between treatment outcomes were calculated using the Mann-Whitney test and between subgroups using the Kruskal-Wallis test with Dunn’s multiple comparisons test. CD107a, Fc-mediated natural killer cell degranulation; IFNγ, Fc-mediated natural killer IFNγ production; ADRB, antibody-dependent respiratory burst by neutrophils; OPA, opsonic phagocytosis activity of MSP1 FL -coupled microsphere beads by monocytes; AbC’, antibody-dependent complement fixation activity.

Article Snippet: The Plasmid containing the codon-optimized sequence of full-length MSP1 (3D7) was received from the plasmid repository Addgene (#47709; Plasmid) which has been optimized for expression in mammalian cells ( ; ).

Techniques: Functional Assay, Activity Assay, Clinical Proteomics, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: Full-length MSP1 is a major target of protective immunity after controlled human malaria infection

doi: 10.26508/lsa.202301910

Figure Lengend Snippet: (A) A heatmap with correlation matrix showing spearman rank correlation coefficients for antibody levels and Fc-mediated effector functions. The color intensity represents the strength of correlation. (B) Forest plot showing adjusted hazard ratios (aHR) for MSP1 FL -specific functional activities ranked from lowest to highest. The aHRs were calculated using the cox regression model comparing the time to treatment between high versus low responders based on function-specific thresholds when adjusting for confounders (drug levels and year of study). Error bars indicate 95% confidence intervals and the red line indicates no protection (aHR = 1.0). CD107a, Fc-mediated natural killer cell degranulation; IFNγ, Fc-mediated natural killer IFNγ production; ADRB, antibody-dependent respiratory burst by neutrophils; OPA, opsonic phagocytosis activity of MSP1 FL -coupled microsphere beads by monocytes; AbC’, antibody-dependent complement fixation activity.

Article Snippet: The Plasmid containing the codon-optimized sequence of full-length MSP1 (3D7) was received from the plasmid repository Addgene (#47709; Plasmid) which has been optimized for expression in mammalian cells ( ; ).

Techniques: Functional Assay, Activity Assay

Journal: Life Science Alliance

Article Title: Full-length MSP1 is a major target of protective immunity after controlled human malaria infection

doi: 10.26508/lsa.202301910

Figure Lengend Snippet: (A) A heatmap showing the activity levels of all five Fc-mediated effector functions in treated (n = 56) and non-treated (n = 86) volunteers. Responses above a function-specific threshold are shown in red. Each column is a Fc-mediated function whereas each row is a single volunteer. AbC’, antibody-dependent complement fixation activity; OPA, opsonic phagocytosis activity of MSP1 FL -coupled microsphere beads by monocytes; ADRB, antibody-dependent respiratory burst by neutrophils; IFNγ, Fc-mediated natural killer IFNγ production; CD107a, Fc-mediated natural killer cell degranulation. (B) Kaplan-Meier plot of volunteers who remained non-treated at different timepoints over the course of 21 d post challenge. Each line represents a function breadth score starting from 0 (n = 15), 1–2 (n = 19), 3–4 (n = 25), and 5 (n = 83). Significance was assessed using the Log rank sum test. (C) Anti-MSP1 FL IgG levels were compared between individuals with different levels of MSP1 FL -specific breadth of function. Each dot represents one sample in duplicates measured in one experiment. Error bars represent the median plus 95% confidence intervals. The seropositivity cut-off value was calculated as the optical density (OD) of malaria-naïve plasma samples plus three standard deviations indicated as the dotted line. Statistical differences were calculated using the Kruskal-Wallis test.

Article Snippet: The Plasmid containing the codon-optimized sequence of full-length MSP1 (3D7) was received from the plasmid repository Addgene (#47709; Plasmid) which has been optimized for expression in mammalian cells ( ; ).

Techniques: Activity Assay, Clinical Proteomics