Human adenovirus 5-vectored Plasmodium falciparum NMRC-M3V-Ad-PfCA vaccine encoding CSP and AMA1 is safe, well-tolerated and immunogenic but does not protect against controlled human malaria infection

Abstract

Background: In a prior study, a DNA prime / adenovirus boost vaccine (DNA/Ad) expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) (NMRC-M3V-D/Ad-PfCA Vaccine) induced 27% protection against controlled human malaria infection (CHMI). To investigate the contribution of DNA priming, we tested the efficacy of adenovirus vaccine alone (NMRC-M3V-Ad-PfCA ) in a Phase 1 clinical trial.

Methodology/principal findings: The regimen was a single intramuscular injection with two non-replicating human serotype 5 adenovectors encoding CSP and AMA1, respectively. One x 10 (10) particle units of each construct were combined prior to administration. The regimen was safe and well-tolerated. Four weeks later, 18 study subjects received P. falciparum CHMI administered by mosquito bite. None were fully protected although one showed delayed onset of parasitemia. Antibody responses were low, with geometric mean CSP ELISA titer of 381 (range<50-1626) and AMA1 ELISA of 4.95 µg/mL (range 0.2-38). Summed ex vivo IFN-γ ELISpot responses to overlapping peptides were robust, with geometric mean spot forming cells/million peripheral blood mononuclear cells [sfc/m] for CSP of 273 (range 38-2550) and for AMA1 of 1303 (range 435-4594). CD4+ and CD8+ T cell IFN-γ responses to CSP were positive by flow cytometry in 25% and 56% of the research subjects, respectively, and to AMA1 in 94% and 100%, respectively.

Significance: In contrast to DNA/Ad, Ad alone did not protect against CHMI despite inducing broad, cell-mediated immunity, indicating that DNA priming is required for protection by the adenovirus-vectored vaccine. ClinicalTrials.gov Identifier: NCT00392015.

Trial registration: ClinicalTrials.gov NCT00392015 NCT00870987.

Keywords: T cells; adenovirus; antibody; efficacy; malaria; vaccine.

Figures

Figure 1. Schematic of Adenovirus CSP and AMA1 vaccines. Each panel presents the native protein (top of each panel) and the protein expressed by the Ad construct (bottom of each panel) for the CSP (A) and AMA1 (B) vaccine antigens. N = N-terminus; C = carboxy terminus; TM = transmembrane domain. Identical colors indicate identical sequences.

Figure 2. Trial design. Subjects were immunized week 0 and challenged week 4. Samples for measuring cell-mediated immunity (ELISpot assay and flow cytometry) and antibody levels (ELISA and IFA) were collected at six time points (black arrows): Pre (pre-immunization), Post-Ad (*22–23 d after immunization), Post-Ch+4 (four weeks after challenge), Post-Ch+12 (12 weeks after challenge), Post-Ch+20 (20 weeks after challenge), and Post-Ch+48 (48 weeks after challenge).

Figure 3. Flow diagram of immunized and control volunteers. Thirty-seven volunteers met all eligibility criteria of whom 11 withdrew consent, and 26 volunteers were allocated to the immunization group (n = 20) and infectivity controls (n = 6) respectively. Two immunized volunteers were not challenged due to family reasons and poor compliance, respectively. Approximately two years after challenge, an infectivity control died of causes unrelated to the vaccine.

Figure 4. Development of parasitemia in the immunized and infectivity volunteers. Parasitemia-free survival curves (Kaplan-Meier) for immunized volunteers and infectivity controls based on microscopic examination of peripheral blood smears.

Figure 5. Antibody responses by ELISA to CSP and AMA1. Group geomean CSP and AMA1 ELISA activities for the 18 recipients were significantly higher than baseline (*) CSP: Post-Ad (CSP p = < 0.0001), Post-Ch+4 CSP (p = < 0.0001), Post-Ch+12 (CSP p = < 0.0001) and Post-Ch+20 (CSP p = 0.0003); AMA1: Post-Ad (p = < 0.0001), Post-Ch+4 CSP (p = < 0.0001), Post-Ch+12 (p = < 0.0001) and Post-Ch+20 (p = < 0.0001). For explanation of box plots (including outliers) see statistics section at the end of Methods below.

Figure 6. Antibody responses by IFA to P. falciparum sporozoites and asexual blood stages. Group geomean IFA activities were significantly higher than baseline (*): Sporozoites: Post-Ad (p = 0. < 0001), Post-Ch+4 (p = 0. < 0001), Post-Ch+12 (p = 0.0015), Post-Ch+20 (p = 0.0002); red blood stages: Post-Ad (p = < 0.0001), Post-Ch+4 (p = < 0.0001), and Post-Ch+12 (p = < 0.0001) and Post-Ch+20 (p = < 0.0001) (mixed linear model). For explanation of box plots (including outliers) see statistics section at the end of Methods below.

Figure 7. Ex vivo T cell IFN-γ activities by ELISpot Assay for CSP and AMA1. Group geomean IFA activities were significantly higher than baseline (*): CSP: Post-Ad (p = < 0.0001), Post-Ch+4 (p = < 0.0001), Post-Ch+12 (p = 0.0083), Post-Ch+20 (p = 0.0009) Post Ch+48 (p = < 0.0001; AMA1 Post-Ad (p = < 0.0001), Post-Ch+4 (p = < 0.0001), Post-Ch+12 (p = 0.0056), Post-Ch+20 (p = 0.0019), Post Ch+48 (p = < 0.0013). For explanation of box plots (including outliers) see statistics section at the end of Methods below.

Figure 8. CD4+ and CD8+ T cell IFN-γ activities by flow cytometry for CSP and AMA1. The box plots represent IFN-γ-producing CD4+ or CD8+ T cell frequencies as percentage of gated CD4+ or CD8+ T cells, measured by flow cytometry assays after stimulation with a single CSP or AMA1 megapool containing all individual peptide pools for each antigen. Arrow denotes v156 who had very high CD8+ T cell activity to AMA1 (2.08%) at Post-Ad. IFN-γ-producing CD4+ T cell activities for CSP were only significantly higher than baseline (*) at Post-Ad (p = 0.0017); CD4+ T cell activities for AMA1 were significantly higher than baseline (*) Post-Ad (p = 0.0002), Post-Ch+4 (p = 0.0017), Post-Ch+20 (p = 0.0054) and Post-Ch+48 (p = 0.0035). IFN-γ-producing CD8+ T cell activities for CSP were only marginally higher than baseline (*) at Post Ad (p = 0.07); CD8+ T cell activities for AMA1 were significantly higher than baseline (*) at Post-Ad (p = 0.0021) and Post-Ch+4 (p = 0.017). For explanation of box plots (including outliers) see statistics section at the end of Methods below.