6 research outputs found

    rCSP Strain Selection and Identification.

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    <p>(<b>A</b>) Reduced SDS-PAGE analysis of lysate samples prior to purification. Lanes 1–3 represent samples from strain CSP-1, CSP-3, and CSP-2 fermentations, respectively. Lane 4 represents a sample from a culture of <i>P. fluorescens</i> transformed with plasmid vector containing no insert. Arrow denotes rCSP in lysate samples. (<b>B</b>) Reduced SDS-PAGE analysis of purified proteins from a 2-column purification process. Lanes 1–3 represent samples from CSP-1, CSP-2, and CSP-3 productions, respectively. (<b>C</b>) Western blot of non-reduced purified CSP-1, CSP-2, and CSP-3 proteins (lanes 2–4, respectively). Lane 1 represents a sample from a culture of <i>P. fluorescens</i> transformed with plasmid vector containing no insert.</p

    Activity of Prime-Boost Regimens in Mice.

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    <p>C57BL/6 mice (5–7 per cohort) were immunized 3x with 20 µg of rCSP formulated in GLA-SE, 1–2×10<sup>10</sup> v.p. of Ad35CS, or a combination of the two. Spleens and livers were collected 10 days after the last immunization; sera were collected 10 or 14 days after the last immunization. To assess reduction in parasite load in the liver or sterile protection, mice were challenged with <i>Pb</i>-CS(<i>Pf</i>) parasites 3 weeks after the last immunization. (<b>A</b>) Percent inhibition of LS parasite development in immunized mice challenged with <i>Pb</i>-CS(<i>Pf</i>) sporozoites normalized to naïve mice received the challenge, and percent sterile protection in immunized BALB/c mice upon challenge with a fewer number of Pb-CS(Pf) sporozoites are shown. A statistically significant reduction of LS parasites in the livers of mice immunized with all regimens, except Ad35CS 3x and Ad35CS (1x), GLA-SE (2x), was seen upon sporozoites challenge, compared to the level of LS parasites in the livers of negative control mice following sporozoites challenge, based on the mean parasite's 18s rRNA copy number (as noted by asterisks). Three regimens were assessed in two independent studies (GLA-SE 3x; GLA-SE 1x, Ad35CS 2x; and Ad35CS 1x, GLA-SE 2x); for these regimens, average percent inhibition is shown with error bars represented as standard error. Statistical significance with the GLA-SE 3x; GLA-SE 1x, Ad35CS 2x was seen in both studies. (<b>B</b>) ELISA was used to evaluate the level of humoral response in immunized mice to the repeat region of CSP with pooled mouse sera and to whole rCSP with individual mouse sera. Titer was assessed at OD = 1.0 based on 4-parameter logistic curve fits. Average titers are shown with error bars representing standard error of the mean. (<b>C</b>) The level of T-cell response was determined via ELISpot using lymphocytes isolated from the spleens and livers of immunized mice (5 mice per cohort were used for this study) and overlapping peptides (15-mers encompassing the CSP repeat region) for in vitro restimulation. Average numbers of IFN-γ producing cells/10<sup>6</sup> lymphocytes are shown with error bars representing standard error of the mean. No more than 5 IFN-γ producing cells/10<sup>6</sup> lymphocytes were counted with cells harvested from naïve mice (data not shown).</p

    Immunogenicity and Efficacy of rCSP Immunization in Mice.

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    <p>Five C57BL/6 mice per cohort were immunized 3x with 25 µg of rCSP from 3 production strains (CSP-1, CSP-2, and CSP-3) formulated in incomplete Freund's adjuvant. Sera were collected 2 weeks after the last immunization and pooled for analysis by ELISA, IFA, and ISI. Mice were challenged with <i>Pb</i>-CS(<i>Pf</i>) parasites 3 weeks after the last immunization. (<b>A</b>) Pooled sera from mice immunized with the 3 recombinant proteins showed similar ELISA and IFA titers. ELISA titers to a [NANP]<sub>6</sub> peptide were calculated at OD = 1.0 based on 4-parameter logistic curve fits, and IFA titers were determined based on the lowest serum dilution to give sporozoites-specific fluorescence above background level shown by sera from naïve mice. (<b>B</b>) Sera from rCSP-immunized mice reacted to <i>Pb</i>-CS(<i>Pf</i>) sporozoites demonstrated the expected pattern of surface fluorescence by IFA. (<b>C</b>) Sera from rCSP-immunized mice diluted 1∶100 and assayed for the ability to block <i>Pf</i> sporozoite infection of hepatocytes in an ISI assay demonstrated the highest ISI activity for anti-CSP-1 protein sera. The percent inhibition is shown relative to the number of sporozoites after incubating with the sera of naïve mice. Percent inhibition of LS parasite development in rCSP-immunized mice challenged with <i>Pb</i>-CS(<i>Pf</i>) sporozoites normalized to naïve control mice is also shown. The reduction of LS parasites in livers of mice immunized with CSP-1 or CSP-3 proteins was statistically significant based on the mean parasite-specific 18 s rRNA copy number compared the level of LS parasites in livers of naïve and adjuvant-only administered mice (as noted by asterisks).</p

    Sterile Protection with rCSP.

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    <p>BALB/c mice were immunized 3x with 25 µg of rCSP formulated in GLA-SE or GLA-LSQ, followed by challenge with Pb-CS(Pf) parasites 2 weeks after the last immunization. Parasitemia was monitored by Giemsa-stained blood smears daily from 7 to 11 days post challenge. Animals with no detectable parasitemia on all days monitored were considered sterilely protected.</p><p>Sterile Protection with rCSP.</p

    Comparison of Humoral Responses with rCSP Adjuvant Formulations in Mice.

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    <p>C57BL/6 mice were immunized 3x with 2.5 µg, 5 µg, or 20 µg of rCSP (CSP-1) formulated in different adjuvants. (<b>A</b>) Pooled sera (7 mice per cohort) collected 2 weeks after the final immunization from mice immunized with GLA-SE, GLA-LSQ and incomplete Freund's adjuvant showed similar ELISA titers to a [NANP]<sub>6</sub> peptide at OD = 1.0 based on 4-parameter logistic curve fits. The titers of the sera from mice injected with rCSP formulated in alum-based adjuvants were lower. The immunogenicity of GLA-SE and GLA-LSQ formulations with 20 µg rCSP was assessed in multiple independent studies (5–7 mice per cohort were used for each study); average titer is shown with error bars represented as standard error. (<b>B</b>) Pooled sera (7 mice per cohort) collected 2 weeks after the final immunization from mice immunized with GLA-SE, GLA-LSQ and incomplete Freund's adjuvant demonstrated the highest IFA endpoint titers based on the lowest serum dilution to give <i>Pb</i>-CS(<i>Pf</i>) sporozoite surface fluorescence above negative sera control level. The titers of the sera from mice immunized with rCSP formulated in alum-based adjuvants were lower. The immunogenicity of GLA-SE and GLA-LSQ formulations with 20 µg rCSP were assessed in multiple independent studies (5–7 mice per cohort were used for each study); average endpoint titer is shown with error bars represented as standard error. (<b>C</b>) Individual immune mouse sera (10 mice per cohort) evaluated in a long-range time course study demonstrated the highest peak ELISA titer to rCSP with the GLA-LSQ formulation ∼8 weeks after the final immunization with a 2.5 µg of rCSP administered per dose for each formulation tested. Average titer at OD = 1 is shown with error bars represented as standard error. Note that a standard scale (rather than log scale) is shown for the y-axis.</p
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