A three-antigen Plasmodium falciparum DNA prime-Adenovirus boost malaria vaccine regimen is superior to a two-antigen regimen and protects against controlled human malaria infection in healthy malaria-naïve adults

Abstract

Background: A DNA-prime/human adenovirus serotype 5 (HuAd5) boost vaccine encoding Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) and Pf apical membrane antigen-1 (PfAMA1), elicited protection in 4/15 (27%) of subjects against controlled human malaria infection (CHMI) that was statistically associated with CD8+ T cell responses. Subjects with high level pre-existing immunity to HuAd5 were not protected, suggesting an adverse effect on vaccine efficacy (VE). We replaced HuAd5 with chimpanzee adenovirus 63 (ChAd63), and repeated the study, assessing both the two-antigen (CSP, AMA1 = CA) vaccine, and a novel three-antigen (CSP, AMA1, ME-TRAP = CAT) vaccine that included a third pre-erythrocytic stage antigen [malaria multiple epitopes (ME) fused to the Pf thrombospondin-related adhesive protein (TRAP)] to potentially enhance protection.

Methodology: This was an open label, randomized Phase 1 trial, assessing safety, tolerability, and VE against CHMI in healthy, malaria naïve adults. Forty subjects (20 each group) were to receive three monthly CA or CAT DNA priming immunizations, followed by corresponding ChAd63 boost four months later. Four weeks after the boost, immunized subjects and 12 infectivity controls underwent CHMI by mosquito bite using the Pf3D7 strain. VE was assessed by determining the differences in time to parasitemia as detected by thick blood smears up to 28-days post CHMI and utilizing the log rank test, and by calculating the risk ratio of each treatment group and subtracting from 1, with significance calculated by the Cochran-Mantel-Haenszel method.

Results: In both groups, systemic adverse events (AEs) were significantly higher after the ChAd63 boost than DNA immunizations. Eleven of 12 infectivity controls developed parasitemia (mean 11.7 days). In the CA group, 15 of 16 (93.8%) immunized subjects developed parasitemia (mean 12.0 days). In the CAT group, 11 of 16 (63.8%) immunized subjects developed parasitemia (mean 13.0 days), indicating significant protection by log rank test compared to infectivity controls (p = 0.0406) and the CA group (p = 0.0229). VE (1 minus the risk ratio) in the CAT group was 25% compared to -2% in the CA group. The CA and CAT vaccines induced robust humoral (ELISA antibodies against CSP, AMA1 and TRAP, and IFA responses against sporozoites and Pf3D7 blood stages), and cellular responses (IFN-γ FluoroSpot responses to CSP, AMA1 and TRAP) that were not associated with protection.

Conclusions: This study demonstrated that the ChAd63 CAT vaccine exhibited significant protective efficacy, and confirmed protection was afforded by adding a third antigen (T) to a two-antigen (CA) formulation to achieve increased VE. Although the ChAd63-CAT vaccine was associated with increased frequencies of systemic AEs compared to the CA vaccine and, historically, compared to the HuAd5 vectored malaria vaccine encoding CSP and AMA1, they were transient and associated with increased vector dosing.

Fig 1. Flow diagram of immunized and control subjects.

Fifty-two subjects met all eligibility criteria and were randomly allocated to the CA group (n = 20), CAT group (n = 20) and infectivity controls (n = 12). CA group: Prior to the ChAd63 immunization, 1 subject withdrew for personal reasons; prior to CHMI, 3 subjects were withdrawn: 1 due to a laboratory abnormality thought possibly related to the study interventions, 1 due to relocation, and 1 due to suicidal ideation requiring inpatient admission (unrelated to the study intervention). CAT group: Prior to the third DNA immunizations, 2 subjects withdrew: 1 subject due to an unrelated SAE and 1 subject due to a medical issue (commencing latent tuberculosis treatment); prior to ChAd63 immunizations, 2 subjects withdrew, 1 subject due to pregnancy, and 1 subject due to a SAE grade 4 laboratory abnormality (neutropenia) likely attributed to benign ethnic neutropenia but possibly to study intervention.

Fig 2. CA and CAT groups: Post-DNA and post-ChAd63 local and systemic adverse events.

Horizontal bars represent percentage of subjects affected by Grade 1, 2 or 3 AEs after DNA and ChAd63 immunizations. This was calculated by dividing the total occurrences of each AE after all 3 DNA immunizations, or after the ChAd63 immunization by the total numbers of subjects that received each immunization: CA group: 60 subjects received three DNA immunizations, and 19 subjects received one ChAd63 immunization; CAT group: 58 subjects received 3 DNA immunizations, and 16 subjects received one ChAd63 immunization. Most adverse events (AEs) were mild to moderate and were similar after each DNA immunization but were more frequent after ChAd63 immunization, especially in the CAT group. Grade 3 AEs in the CA group after ChAd63 were one each of fatigue, nausea, and vomiting. However, in the CAT group Grade 3 AEs were more frequent.

Fig 3. Kaplan-Meier curve (CAT and CA).

K-M curves depicting the CAT and CA groups.

Fig 4. Kaplan-Meier curve [CAT and infectivity controls (IC)].

K-M curves depicting the CAT and infectivity controls (IC) group. In both K-M curves data was censored at day 28. Parasitemia was based on microscopic examination of peripheral blood smears.

Fig 5. CA and CAT cohorts: ELISA antibody responses to CSP.

The box plots (see Statistical Analysis section for description) represent anti-CSP by ELISA for all challenged subjects. The time points on the x-axis are described in Methods. Protected subjects are shown as larger, color-coded dots.

Fig 6. CA and CAT cohorts: ELISA antibody responses to AMA1 and TRAP.

The box plots (see Statistical Analysis section for description) represent anti-AMA1 titers and anti-TRAP activities by ELISA for all challenged subjects. The time points on the x-axis are described in Methods. Protected subjects are shown as larger, color-coded dots.

Fig 7. Antibody responses by IFA to PfNF54 sporozoites and Pf3D7 blood stages.

The box plots represent responses to PfNF54 sporozoites and Pf3D7 blood stages. Responses of protected subjects are color-coded; not protected (NP) subjects are open circles.

Fig 8. CA cohort: IFN-γ FluoroSpot responses to CSP and AMA1.

Ex-vivo T-cell activities by FluoroSpot Assay for CSP and AMA1. The box plots represent CSP and AMA1. Summed IFN-γ T-cell responses against peptide pools are spot forming cells per million PBMCs for all challenged subjects.

Fig 9. CAT cohort: IFN-γ FluoroSpot responses to CSP, AMA1 and TRAP.

Ex-vivo T-cell activities by FluoroSpot Assay for CSP, AMA1 and TRAP. The box plots represent CSP, AMA1 and TRAP (Pf9/96 and Pf3D7). Summed IFN-γ T-cell responses against peptide pools are spot forming cells per million PBMCs for all challenged subjects.