Volume 5, Issue 1, Pages 95-105 (January 2009) Recombinant Viral Vaccines Expressing Merozoite Surface Protein-1 Induce Antibody- and T Cell-Mediated Multistage Protection against Malaria  Simon J. Draper, Anna L. Goodman, Sumi Biswas, Emily K. Forbes, Anne C. Moore, Sarah C. Gilbert, Adrian V.S. Hill  Cell Host & Microbe  Volume 5, Issue 1, Pages 95-105 (January 2009) DOI: 10.1016/j.chom.2008.12.004 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Immunogenicity of AdHu5-MVA-MSP-1 Recombinants (A and B) BALB/c mice were primed i.d. with AdHu5 expressing MSP-142, MSP-133, or MSP-119 (Ad42, Ad33, or Ad19 respectively) and boosted i.d. 56 days later with the corresponding MVA vector (M42, M33, or M19). Fourteen days after boost, splenocytes were restimulated with MSP-133 peptides, and the production of IFN-γ, TNF-α, and IL-2 by CD4+ (A) and CD8+ (B) T cells in the spleen was assessed using polychromatic flow cytometry. The functional compositions of the CD4+ and CD8+ T cell responses are shown, and these are grouped and color-coded according to the vaccine regime and number of cytokines secreted by each T cell population. The pie charts in (B) summarize the fractions of MSP-133-specific CD8+ T cells that are positive for a given number of functions. Individual data points and mean percentage of the total response (open bars) are shown for each of the functional populations indicated on the x axis. No responses were detected above background following restimulation with MSP-119 peptides (data not shown). Representative flow cytometry plots and the gating strategy are shown in Figure S1. (C and D) Total IgG serum antibody responses against MSP-119 and MSP-133 were measured by ELISA in mice primed and boosted as indicated. Mean responses ± SEM are shown (n = six mice per group). N.D. = not detected. ∗ p ≤ 0.05 in (C) (independent Student's t test comparing responses between the AdM42 and AdM33 groups). Similar results were obtained in three independent experiments. Cell Host & Microbe 2009 5, 95-105DOI: (10.1016/j.chom.2008.12.004) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Survival against pRBC and Sporozoite Challenge (A–C) BALB/c mice were immunized as indicated. Fourteen days after the final immunization, these mice or naive controls were challenged i.v. with 104 pRBCs (A) or 50 sporozoites (B), and Kaplan-Meier plots show survival over time. Data were pooled from multiple experiments, and the number of mice challenged in total (n) is indicated for each group. As shown in (C), mice immunized with AdM33 or naive controls were challenged with 104 pRBCs or 50 sporozoites. Parasitemia in the blood was measured postchallenge by microscopy. Plots show the mean parasitemia ± SEM (n = six mice per group) for the first 4 days following patency. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001 (independent Student's t test comparing percent parasitemia at each time point between the immunized and naive control mice). Cell Host & Microbe 2009 5, 95-105DOI: (10.1016/j.chom.2008.12.004) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 AdHu5-MVA-MSP-1 Immunization Efficacy against Liver-Stage Infection (A–E) BALB/c mice were immunized with AdM42 or AdM42-C4bp (A), AdM42-C4bp (B), or AdM33 or AdM-GFP vector controls (C), and challenged with 5000 sporozoites 14 days after the boost. Liver-stage parasite burden was quantified 48 hr postchallenge using an established real-time RT-PCR assay (Bruna-Romero et al., 2001). In (B), mice immunized with AdM42-C4bp were depleted of CD8+ or CD4+ T cells or control depleted with normal rat IgG (nRat IgG) prior to challenge. (D) shows blood-stage parasitemia on day 5 in BALB/c mice immunized as indicated or immunized with AdM33 and depleted of T cell subsets as indicated (E) and challenged with 50 sporozoites. The mean ratio or parasitemia ± SEM is shown (n = five or six mice per group). ∗ p ≤ 0.05 and ∗∗ p ≤ 0.01, comparing between all groups by one-way ANOVA. Cell Host & Microbe 2009 5, 95-105DOI: (10.1016/j.chom.2008.12.004) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Blood-Stage Parasitemias in Naive and Immunized Mice 48 hr after Blood-Stage Infection (A) BALB/c mice immunized with AdM33 were challenged with 50 sporozoites (n = 18), or naive controls were challenged with 104 pRBCs (n = 29), 50 sporozoites (n = 44), or 250 sporozoites (n = 8). Blood-stage parasitemia ∼48 hr post blood-stage infection was assessed by microscopy (day 5 post spz challenge and day 2 post pRBC challenge). Data were pooled from multiple experiments. Plots show median values, 25th–75th percentiles, and range. One mouse in the AdM33 group had 0% parasitemia on day 5. (B) BALB/c mice were immunized with AdM33 or 1 × 108 vp AdHu5-PyCSP and challenged with 50 sporozoites 14 days later. The mean parasitemia ± SEM is shown (n = five or six mice per group). ∗p ≤ 0.05 and ∗∗∗ p ≤ 0.001, comparing between all groups by one-way ANOVA. Cell Host & Microbe 2009 5, 95-105DOI: (10.1016/j.chom.2008.12.004) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Serum Cytokine Responses Following pRBC and Sporozoite Challenge (A–F) BALB/c mice were immunized as indicated and challenged with 104 pRBCs (A, C, and E) or 50 sporozoites (B, D, and F) or not challenged (“AdM33 control”) (A, C, and E). Serum was collected prechallenge (0 hr), 48 hr post blood-stage infection (day 5 post spz challenge and day 2 post pRBC challenge), and 120 hr post blood-stage infection (day 8 post spz challenge and day 5 post pRBC challenge). Serum cytokine levels were assayed by flow cytometry using a cytometric bead array assay. Mean levels ± SEM are shown (n = five or six mice per group) for IFN-γ (A and B), IL-10 (C and D), and TNF-α (E and F). ∗ p ≤ 0.05, comparing between groups (independent Student's t test in [A] and [C]), and one-way ANOVA in (B). Similar results were obtained in two independent experiments. Cell Host & Microbe 2009 5, 95-105DOI: (10.1016/j.chom.2008.12.004) Copyright © 2009 Elsevier Inc. Terms and Conditions