Qβ-CSP <i>vs</i>. CSP in Montanide.

<p>Groups of 6 mice were vaccinated thrice with 2.5, 1 and 0.1 μg CSP or Qβ-CSP in Montanide. A, B show the individual data points and mean±SEM titers against full-length protein (A) and NANP repeat peptide (B) 2 weeks post 3^rd vaccination (2WP3). **** (p<0.0001 for ANOVA followed by Tukey’s multiple comparisons test); red data points correspond to mice protected 14 days post challenge and numbers (blue) were protected out of 6. C, Immunofluorescence image of methanol fixed sporozoites stained with 1:2500 dilution of anti-CSP pool (left) or Qβ-CSP serum pool (right) for the 1 μg dose groups. D, E show IgG1, IgG2b and IgG2c levels measured by Luminex and expressed as median fluorescence intensity (MFI) at 1:1000 serum dilution against the NANP peptide (D) or the C-term protein (E).</p

Qβ-CSP <i>vs</i>. CSP in SE, GLA/SE and Alum.

<p>Groups of 10 mice received 3 doses, 3 weeks apart of 2.5 μg CSP+SE, Qβ-CSP+SE, CSP+GLA/SE, Qβ-CSP+GLA/SE or Qβ-CSP+Alum. A, B show the mean±SEM of the full-length and NANP-specific ELISA titers at 2 weeks post third vaccination. ** (p<0.01); * (p<0.05); red symbols represent protected mice and number (blue) protected out of 10. C, D, E and F are data from an independent 2-dose study. 15 mice received 2 doses of 2.5 μg CSP+GLA/SE and Qβ-CSP+GLA/SE, three weeks apart. C, D show the mean±SEM of full-length and NANP-specific ELISA titer from the 10 challenged mice at 2 weeks after the second vaccination. Red symbols were protected mice and number represent protected out of 10 (blue). E, F, IgG1, IgG2b and IgG2c responses of 15 mice measured by Luminex and expressed as MFI at 1:2000 dilution against NANP peptide (E) or C-term protein (F).</p

Combined ELISA titer and protection data of CSP <i>vs</i>. Qβ-CSP groups.

<p>Full-length (left) and NANP (right) titers were plotted for individual animals in Montanide, Alum and SE adjuvanted CSP and Qβ-CSP groups. Combined protection data is indicated (blue). Lines are mean±SEM and the P values are for unpaired T test performed on log transformed titers.</p

Qβ-CSP <i>vs</i>. CSP in Alum.

<p>Groups of 10 mice received 2 doses, 3 weeks apart of 2.5 μg CSP+Alum, Qβ-CSP+Alum or Qβ-CSP without an adjuvant. A, B show the mean±SEM of the full-length and NANP-specific ELISA titers at 2 weeks after the second vaccination. **** (p<0.0001); *** (p<0.001); * (p<0.05); red symbols represent protected mice and number (blue) protected out of 10. C, D, show the IgG1, IgG2b and IgG2c responses measured by Luminex and expressed as MFI at 1:1000 dilution against NANP peptide (C) or C-term protein (D).</p

Characterization of Qβ-CSP vaccine.

<p>A, identity of the product bands was confirmed using reducing coomassie blue staining and western blot with polyclonal mouse anti-Qβ and anti-CSP antibodies. Lanes 1, 2, Qβ-CSP; lane 3, unconjugated Qβ; lane 4, unconjugated CSP. B, particle size distribution of Qβ-CSP by dynamic light scatter analysis. C, electron micrograph of the negatively stained Qβ-CSP. D, immunological reactivity of the soluble CSP and Qβ-CSP against CSP-specific mAbs targeting the NANP repeats, the C-term region or epitopes present only on full-length (FL) CSP. Mabs labelled as “Hu” were produced in humanized mice and “Mo” were produced in wild-type mice.</p

Production of the Qβ-CSP vaccine.

<p>A, Outline of the conjugation process. Numbers in parentheses correspond to the respective lane number in Fig 1B. B, CSP and Qβ proteins analyzed by reducing SDS-PAGE followed by coomassie blue staining (left) or western blot using anti-CSP polyclonal mouse antibodies (right). Lane 1, soluble CSP protein; 2, CSP diluted in urea; 3, SATA treated and desalted CSP; 4, Qβ protein; 5, SMPH treated Qβ; 6, desalted Qβ-Malemide; 7, deacetylated and desalted CSP; 8, CSP-Qβ conjugate; 9, desalted CSP-Qβ conjugate (final vaccine); M, molecular weight marker.</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

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