Fig. 2: Immunogenicity following intranasal priming and oral boosting.

a Study design. On Day 0, BALB/cJ mice were primed via one of three ways: 1. Orally (PO) with unpurified spirulina biomass resuspended in HBSS; 2. Intranasally (IN) with unpurified spirulina biomass resuspended in PBS; or 3. Intranasally with spirulina total soluble protein extract. All doses were supplemented with Montanide adjuvant. Oral Montanide-adjuvanted spirulina booster doses (containing control or PfCSP unpurified biomass) were then given 1–3 times on Days 28, 42, and up to 56. Subcutaneous challenge with 2 × 10³ wild-type PfCSP-expressing P. yoelii spz was conducted two weeks after the last booster. Serum was collected where indicated by large drops. Protection was assessed by blood smears (small drops). Endpoint titer ELISA results for PfCSP antibodies are shown two weeks after priming (b, Day 14) or two weeks after the third booster (c, Day 70). PfCSP-PyRAS (x3) refers to replicate wells of pooled sera obtained from female BALB/cJ mice immunized three times with irradiated Py spz expressing PfCSP as a benchmark control. Groups are as shown in the legend below each dataset; each data point is an individual mouse. Error bars show mean and standard deviation. *p < 0.05; **p < 0.01; ***p*<0.0001; ns not significant p > 0.05 (ANOVA with post-hoc Tukey test for multiple comparisons).