Fig 2: T cell responses in mice following mRNA dose de-escalation and delayed boosting.(A) C57BL/6J mice (n=7/grp) were immunized 3x with 25μg, 15μg, and finally 5μg of MIP3α-CSP LNP-mRNA or CSP LNP-mRNA. In this delayed dosing experiment, the 2nd and 3rd immunizations occurred 2 and 8 weeks after the first immunization respectively. Splenocytes were harvested from each mouse 42h after challenge for usage in T cell stimulation assays. (B-C) Cells were stained, run on flow cytometry, and gated for CD4+ (B) or CD8+ (C) T cells. Median fluorescence intensities of CD4+ IFNγ+ (B) and CD8+ IFNγ+ (C) T cells are shown (left). The percentage of IFN-γ+ cells among CD4+ cells (B) and CD8+ cells (C) are also shown (right). Horizontal lines indicate group means. Ordinary one-way ANOVAs with Tukey’s test for multiple comparisons were performed to compare differences between groups. (D-E) Cells were stained, run on flow cytometry, and gated for CD4+ T cells. Median fluorescence intensities of CD4+ TNFα+ cells (D) and CD4+ IL2+ cells (E) are shown (left). The percentage of TNFα+ (D) or IL2+ cells (E) among CD4+ cells are also shown (right). Horizontal lines indicate group means. Ordinary one-way ANOVAs with Tukey’s test for multiple comparisons were performed to compare differences between groups. (F-G) The percentage of CD4+ or CD8+ (G) T cells that were IFNγ, TNFα, and IL-2 triple positive (left axis) or IFNγ, TNFα double positive are shown (right axis). Horizontal lines indicate group means. Ordinary one-way ANOVAs with Tukey’s test for multiple comparisons were performed to compare differences between groups.

Fig 3: Immunological Correlates of Protection.(A) Relationship between log10 NANP6 peptide antibody titers (y-axis) and % inhibition of liver stage parasitemia relative to naïve, sporozoite challenged mice (x-axis). Unfilled and filled symbols represent single mice from challenge studies 1 and 2 (standard regimen and delayed, dose de-escalation regimen) respectively. Squares represent mice from the MIP3α-CSP LNP-mRNA vaccinated group, circles represent mice from the CSP LNP-mRNA vaccinated group, and triangles represent mice from the rCSPFL vaccinated group. P values were calculated using Spearman’s ranked correlation coefficient. (B-D) Relationship between inhibition of liver stage parasitemia (x-axis) and the percentage of IFNγ+ cells among CD4+ cells (B), the percentage of IFNγ, TNFα, and IL-2 triple positive cells among CD4+ cells (C), and the percentage of IFNγ, TNFα, and IL-2 triple positive CD8+ cells (D). Circles represent mice from the CSP vaccinated group, and squares represent mice from the MIP3α-CSP vaccinated group. P values were calculated using

Fig 4: Effect of route of administration and vaccine formulation on T-cell recruitment to the lungs. The vaccination and tissue-harvesting schedule is described in Figure 2A . Three weeks after the final vaccination, the recruitment of T-cell subpopulations to the lungs elicited by the different immunization protocols was determined by flow cytometric analysis, as described in Methods. (A) All CD3+ cells, as well as (B) CD3+CD8+ and (C) CD3+CD4+ cells, were analyzed by normalized cell counts. Differences between groups were analyzed using one-way ANOVA. Six mice per group, three males (open) and three females (filled), with each symbol representing one mouse. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Fig 5: T cell responses in mice following mRNA dose de-escalation and delayed boosting.A C57BL/6J mice (n = 7/grp) were immunized 3x with 25 μg, 15 μg, and finally 5 μg of MIP3α-CSP LNP-mRNA or CSP LNP-mRNA. In this delayed dosing experiment, the 2nd and 3rd immunizations occurred 2 and 8 weeks after the first immunization respectively. Splenocytes were harvested from each mouse 42 h after challenge for usage in T cell stimulation assays. Cells were stained, run on flow cytometry, and gated for CD4+ (B) or CD8+ (C) T cells. Median fluorescence intensities of CD4 + IFNγ + (B) and CD8 + IFNγ + (C) T cells are shown (left). The percentage of IFN-γ+ cells among CD4+ cells (B) and CD8+ cells (C) are also shown (right). Horizontal lines indicate group means. Ordinary one-way ANOVAs with Tukey’s test for multiple comparisons were performed to compare differences between groups. D, E Cells were stained, run on flow cytometry, and gated for CD4 + T cells. Median fluorescence intensities of CD4 + TNFα + cells (D) and CD4 + IL2+ cells (E) are shown (left). The percentage of TNFα + (D) or IL2+ cells (E) among CD4+ cells are also shown (right). Horizontal lines indicate group means. Ordinary one-way ANOVAs with Tukey’s test for multiple comparisons were performed to compare differences between groups. F, G The percentage of CD4+ or CD8+ (G) T cells that were IFNγ, TNFα, and IL-2 triple positive (left axis) or IFNγ, TNFα double positive are shown (right axis). Horizontal lines indicate group means. Ordinary one-way ANOVAs with Tukey’s test for multiple comparisons were performed to compare differences between groups.