Dose response study of CS/D+GLA/SE vaccine in C57Bl/6 mice.

<p>Groups of 7 mice received three immunizations of 0.1, 1, 2.5, 5 or 10 µg CS/D, adjuvanted with GLA/SE at two week intervals. ELISA end-point titers were measured 2 weeks after the last dose against CS/D (left) or NANP (right). Red circles represent protected mice while black circles represent non-protected mice.</p

Immunological responses induced in Balb/c mice by vaccine formulations using SE, Alum or TLR ligands.

<p>Groups of 10 Balb/c mice were immunized with 2 doses of 2.5 µg CSP, 2 months apart in the indicated adjuvant. Two weeks post second immunization, the mice were bled and the serum was analyzed. <i>A,</i> ELISA end point titers of Balb/c mice, measured against CS/D (left) or NANP repeat peptide (right). <i>B,</i> IgG1 (left) and IgG2a (right) subclasses measured by Luminex and expressed as mean fluorescence intensities (MFI) at 1∶500 serum dilution. <i>C,</i> Percentage of total cytokine⁺CD44⁺CD4⁺ T lymphocytes, extracted from vaccinated Balb/c mice and stimulated with CS/D, stained for surface expression of CD3, CD4 and CD44 and intra-cellular expression of IL-2 (left) or IFN-γ (right). Lines are mean with SEM. (*) indicates significant P values for ANOVA followed by Tukey’s multiple comparison test.</p

Recombinant CS/D constructs.

<p><i>A,</i> Cartoon representation of the native <i>P. falciparum</i> CSP consisting of a signal sequence and GPI anchor sequence (red), N-terminal region (green), NVDP and NANP repeats (blue) and a cysteine rich C-terminal region (purple). The expressed constructs (CS/A, CS/C, CS/D and CS/E), their PfCSP-specific start and end residues, and the relative positions of the five cysteine residues (“C”) are shown<b>.</b> <i>B,</i> SDS-PAGE shows relative expression levels (arrows) in un-induced (Un) and induced (In) <i>E. coli</i> cells producing CS/C, CS/D and CS/E.</p

Immunological responses induced in Balb/c mice by CS/D covalently conjugated to 3M-051.

<p>Groups on 10 Balb/c mice were immunized two doses of 2.5 µg CSP, 2 months apart in the indicated adjuvant. One month post second immunization sera were collected and analyzed. <i>A,</i> ELISA end point titers of mice, measured against CS/D (left) or NANP repeat peptide (right). <i>B,</i> IgG1 (left) and IgG2a (right) subclasses expressed as Luminex mean fluorescence intensities (MFI) at 1∶500 serum dilution. <i>C</i>, Percentage of total Cytokine⁺CD44⁺CD4⁺ T lymphocytes, extracted from vaccinated Balb/c mice and stimulated with CS/D, stained for intracellular IL-2 (left) or IFN-γ (middle) or CD8⁺ cells stimulated with the K^d-restricted peptide and stained for IFN-γ (right). Lines are mean with SEM.</p

Immunological responses induced in C57Bl/6 following 2 vaccinations with CS/D adjuvanted with GLA/SE or 3M051.

<p>Groups on 15 C57Bl/6 mice were immunized with 2 doses of 2.5 µg of CSP, 3 weeks apart, in the indicated adjuvant. Two weeks post second immunization sera were analyzed. <i>A,</i> ELISA end point titers of mice, measured against CS/D (left) or NANP repeat peptide (right). Two high responding mice from the 3M051/SE conjugate group were included in statistical analysis but are outside the scale of the NANP graph. <i>B,</i> Relative IgG1 <i>vs.</i> IgG2c responses against the C term protein (left) or NANP peptide (right) measured by Luminex at 1∶2000 serum dilution. <i>C,</i> Percentage of total Cytokine⁺CD44⁺CD4⁺ T lymphocytes, extracted from vaccinated C57Bl/6 mice and stimulated with CS/D, stained for intracellular IL-2 (left) or IFN-γ (right). Lines are mean with SEM.</p

Immunological responses induced in C57Bl/6 following 3 vaccinations with CS/D adjuvanted with GLA/SE or 3M051.

<p>Groups of 15 C57Bl/6 mice were immunized three times, 3 weeks apart, with 2.5 µg of CSP in the indicated adjuvant. Sera were analyzed 2 weeks after the last dose. <i>A,</i> ELISA titers of mice measured against CS/D (left) and NANP peptide (right). Two high responding mice from the GLA/SE group were included in statistical analysis are outside the scale of the NANP graph. <i>B,</i> Relative IgG1 <i>vs.</i> IgG2c responses against the C term protein (left) or NANP peptide (right) measured by Luminex at 1∶2000 serum dilution. C, Percentage of total Cytokine⁺CD44⁺CD4⁺ T lymphocytes, extracted from vaccinated C57Bl/6 mice and stimulated with CS/D, stained for intracellular IL-2 (left) or IFN-γ (right). Lines are mean with SEM.</p

ELISA end-point titers of CSP mAbs.

<p>Full-length CS/D protein, repeat (NANP)₆ peptide and non-reduced or reduced/alkylated (red/alk) version of a C-term peptide (EPSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICKMEKCS) was coated in wells and mAb dilution that resulted in an OD = 1.0 was plotted.</p

Molecular structure of TLR agonists GLA, R848, and CS/D-3M051 conjugate.

<p>Molecular structure of TLR agonists GLA, R848, and CS/D-3M051 conjugate.</p

Protective efficacy in C57Bl/6 mice against transgenic <i>P. berghei</i> sporozoite challenge.

<p><i>A,</i> Survival curves of C56Bl/6 mice (n = 10), challenged with the transgenic parasites in the 2-dose (left) or 3-dose (right) study. Protection was defined as the absence of blood stage infection until day 14 post challenge.</p

Specifications of cGMP-grade CS/D product.

<p>Specifications of cGMP-grade CS/D product.</p