DNA prime/Adenovirus boost malaria vaccine encoding P. falciparum CSP and AMA1 induces sterile protection associated with cell-mediated immunity

Abstract

Background: Gene-based vaccination using prime/boost regimens protects animals and humans against malaria, inducing cell-mediated responses that in animal models target liver stage malaria parasites. We tested a DNA prime/adenovirus boost malaria vaccine in a Phase 1 clinical trial with controlled human malaria infection.

Methodology/principal findings: The vaccine regimen was three monthly doses of two DNA plasmids (DNA) followed four months later by a single boost with two non-replicating human serotype 5 adenovirus vectors (Ad). The constructs encoded genes expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1). The regimen was safe and well-tolerated, with mostly mild adverse events that occurred at the site of injection. Only one AE (diarrhea), possibly related to immunization, was severe (Grade 3), preventing daily activities. Four weeks after the Ad boost, 15 study subjects were challenged with P. falciparum sporozoites by mosquito bite, and four (27%) were sterilely protected. Antibody responses by ELISA rose after Ad boost but were low (CSP geometric mean titer 210, range 44-817; AMA1 geometric mean micrograms/milliliter 11.9, range 1.5-102) and were not associated with protection. Ex vivo IFN-γ ELISpot responses after Ad boost were modest (CSP geometric mean spot forming cells/million peripheral blood mononuclear cells 86, range 13-408; AMA1 348, range 88-1270) and were highest in three protected subjects. ELISpot responses to AMA1 were significantly associated with protection (p = 0.019). Flow cytometry identified predominant IFN-γ mono-secreting CD8+ T cell responses in three protected subjects. No subjects with high pre-existing anti-Ad5 neutralizing antibodies were protected but the association was not statistically significant.

Significance: The DNA/Ad regimen provided the highest sterile immunity achieved against malaria following immunization with a gene-based subunit vaccine (27%). Protection was associated with cell-mediated immunity to AMA1, with CSP probably contributing. Substituting a low seroprevalence vector for Ad5 and supplementing CSP/AMA1 with additional antigens may improve protection.

Trial registration: ClinicalTrials.govNCT00870987.

Conflict of interest statement

Competing Interests: CD and LS from USAID (funders) played a role in study design. JTB and CRK worked for Gen Vec, Inc. (financial interest in the vaccine) and helped design the vaccine constructs. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Trial design.

Subjects were immunized week 0, 4, 8 and 24 and challenged week 28 (blue arrows). Samples for measuring cell-mediated immunity (ELISpot assay and flow cytometry) were collected at six time points (black arrows), and for measuring antibody levels (ELISA, IFA and growth inhibition assay) at similar time points plus after the DNA immunizations (gray arrows). See text for details.

Figure 2. Schematic of DNA and Adenovirus CSP and AMA1 vaccines.

Each panel presents the native protein (top of each panel) and the protein expressed by the DNA or Ad construct (middle and bottom of each panel) for the CSP (Panel A) and AMA1 (Panel B) vaccine antigens. N = amino terminus; C = carboxy terminus; TPA = human tissue plasminogen activator signal sequence; TM = transmembrane domain. See text for explanation. Identical colors indicate identical sequences. Not represented is a single amino acid substitution (G → R) in the AMA DNA construct at position 143.

Figure 3. Flow diagram of immunized and control volunteers.

Thirty-seven volunteers met all eligibility criteria and were allocated to the immunization group (n = 20) and infectivity controls (n = 6), and 11 were either alternates (n = 6) or not used. WBC = white blood count; DVT = deep venous thrombosis. See text for explanation.

Figure 4. Development of parasitemia in the immunized and infectivity control subjects.

Panel A: Parasitemia-free survival curves (Kaplan-Meier) for immunized volunteers and infectivity controls based on microscopic examination of peripheral blood smears. Panel B: Quantitative(q)-PCR measurements of parasitemia in immunized and challenge controls (error bars show standard deviation) (see reference 28).

Figure 5. Pre-existing NAb to Ad5 may interfere with protection.

Pre-existing NAb titers to Ad5 were measured prior to Ad immunization and are compared with days to patency by microscopy after CHMI. Three of six volunteers who were seronegative (NAb titer 500 (above horizontal red line) became patent at a rate similar to subjects with NAb titer

Figure 6. Antibody responses by ELISA to CSP and AMA1.

The box plots (see Statistical Analysis section for description) represent anti-CSP titers and anti-AMA1 antibody concentration in µg/mL by ELISA for all 15 challenged volunteers. The time points on the x-axis are described in Figure 1. Four protected volunteers are shown as larger, color-coded dots. For the protected volunteers, the antibody titer to CSP of v11 post-DNA and the antibody concentration to AMA1 of v11 post-Ad are box plot outliers. Group geomean CSP and AMA1 ELISA activities for the fifteen recipients were significantly higher than baseline (*) post-DNA, post-Ad, post-Ch and post-Ch final relative to pre-immunization levels (p = 

Figure 7. Ex vivo T cell IFN-γ activities by ELISpot Assay for CSP and AMA1.

The box plots (see Statistical Analysis section for description) represent CSP and AMA1 IFN-γ ELISpot responses (summed peptide pool-specific responses) in spot forming cells per million PBMCs for all 15 challenged volunteers. The time points on the x-axis are described in Figure 1. For the protected volunteers, the IFN-γ ELISpot responses to CSP of v11 and v18 post-Ad are box plot suspected outliers. Group geomean CSP and AMA1 IFN-γ ELISpot activities for the fifteen recipients were not significantly higher than baseline post-DNA, pre-Ad, post-Ad, post-Ch or post-Ch final relative to pre-immunization levels (CSP p = 0.057, AMA1 p = 0.16, mixed linear model).

Figure 8. IFN-γ activities by flow cytometry for CSP and AMA1.

The box plots (see Statistical Analysis section for description) 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, for all 15 challenged volunteers. The time points on the x-axis are described in Figure 1. The four protected volunteers are shown as larger, color-coded dots. For the protected volunteers, the CD4+ T cell AMA1 response of v06 at pre-Ad is a box plot outlier, and the CD8+ T cell CSP responses of v11 and v18 post-Ad, and the CD8+ T cell AMA1 responses of v18 post-Ad and post-Ch are box plot suspected outliers. The dotted lines represent positive cutoff (0.03% as described in Methods). IFN-γ -producing CD4+ T cell frequencies were significantly higher than baseline (*) post-DNA (p = 0.047), post-Ad (p = 0.0097) and post-Ch (p = 0.004) for AMA1 (mixed linear model). IFN-γ -producing CD8+ T cell frequencies were significantly higher than baseline (*) post-Ad for CSP (p = 0.007) and post-Ad for AMA1 (0.002) (mixed linear model).