Safety, Immunogenicity, and Protective Efficacy against Controlled Human Malaria Infection of Plasmodium falciparum Sporozoite Vaccine in Tanzanian Adults

Abstract

We are using controlled human malaria infection (CHMI) by direct venous inoculation (DVI) of cryopreserved, infectious Plasmodium falciparum (Pf) sporozoites (SPZ) (PfSPZ Challenge) to try to reduce time and costs of developing PfSPZ Vaccine to prevent malaria in Africa. Immunization with five doses at 0, 4, 8, 12, and 20 weeks of 2.7 × 10⁵ PfSPZ of PfSPZ Vaccine gave 65% vaccine efficacy (VE) at 24 weeks against mosquito bite CHMI in U.S. adults and 52% (time to event) or 29% (proportional) VE over 24 weeks against naturally transmitted Pf in Malian adults. We assessed the identical regimen in Tanzanians for VE against PfSPZ Challenge. Twenty- to thirty-year-old men were randomized to receive five doses normal saline or PfSPZ Vaccine in a double-blind trial. Vaccine efficacy was assessed 3 and 24 weeks later. Adverse events were similar in vaccinees and controls. Antibody responses to Pf circumsporozoite protein were significantly lower than in malaria-naïve Americans, but significantly higher than in Malians. All 18 controls developed Pf parasitemia after CHMI. Four of 20 (20%) vaccinees remained uninfected after 3 week CHMI (P = 0.015 by time to event, P = 0.543 by proportional analysis) and all four (100%) were uninfected after repeat 24 week CHMI (P = 0.005 by proportional, P = 0.004 by time to event analysis). Plasmodium falciparum SPZ Vaccine was safe, well tolerated, and induced durable VE in four subjects. Controlled human malaria infection by DVI of PfSPZ Challenge appeared more stringent over 24 weeks than mosquito bite CHMI in United States or natural exposure in Malian adults, thereby providing a rigorous test of VE in Africa.

Trial registration: ClinicalTrials.gov NCT02132299.

Figure 1.

Volunteer participation (CONSORT 2010 diagram). This figure appears in color at www.ajtmh.org.

Figure 2.

Kaplan–Meier survival curves in immunized volunteers vs. controls as assessed by quantitative polymerase chain reaction (qPCR). Kaplan–Meier curves in volunteers undergoing controlled human malaria infection (CHMI) 3 weeks after the last of five doses with 1.35 × 10⁵ (Group 2) (A) or 2.7 × 10⁵ (Group 3) (B) Plasmodium falciparum Sporozoites (PfSPZ) of PfSPZ Vaccine. Panel (C) volunteers undergoing either first (Group 4) or second (Group 3) CHMI 24 weeks after the fifth immunization with 2.7 × 10⁵ PfSPZ of PfSPZ Vaccine. (D) Vaccine efficacy and prepatent period results. *This was the second CHMI for the 4 volunteers in Group 3 who were protected after the first CHMI at 3 weeks. **One volunteer was inappropriately treated on day 13 for a false positive TBS. Without this volunteer, 3/3 protected. With this volunteer 4/4 were protected. ***Confidence intervals were calculated using Wilson’s score interval. ****Volunteers in CHMI #1 and #2 (3 week CHMI in Groups 2 and 3) had specimens first acquired on day9. Volunteers in CHMI #3 (24 week CHMI in Groups 3 and 4) had specimens first acquired on day 8. This figure appears in color at www.ajtmh.org.

Figure 3.

Antibody responses to Plasmodium falciparum Sporozoites (PfSPZ) and PfCSP before controlled human malaria infection (CHMI). For all assays, uninfected subjects are shown as filled (black) circles and infected subjects are open circles. For each of the defined subject groups, the interquartile ranges and the median values of response of subjects in each group are shown. Assessment of antibodies was performed in sera from subjects before immunization and before CHMI #1 (∼2 weeks after the last dose of PfSPZ Vaccine or normal saline [NS]) and/or CHMI #2 (∼24 weeks after last dose of PfSPZ or NS) (A, D). Antibodies to PfCSP by ELISA are reported as net optical density (OD) 1.0 (the difference in OD 1.0 between pre-CHMI and preimmunization sera). (B, E) Antibodies to PfSPZ by aIFA are reported as net AFU 2 × 10⁵, the reciprocal serum dilution at which the fluorescent units were 2 × 10⁵ (AFU 2 × 10⁵) in pre-CHMI minus preimmunization sera. (C, F) Results of inhibition of sporozoite invasion (ISI) assay are reported as serum dilution at which there was 80% reduction of the number of PfSPZ that invaded a human hepatocyte line (HC-04) in the presence of pre-CHMI as compared with preimmunization sera from the same subject. Panels A–C show groups 2 (five doses of 1.35 × 10⁵ PfSPZ) and 3 (five doses of 2.7 × 10⁵ PfSPZ) before short-term CHMI (2 weeks after the last dose of PfSPZ or NS) and panels D–F show those volunteers in Groups 3 (five doses of 2.7 × 10⁵ PfSPZ) and 4 (five doses of 2.7 × 10⁵ PfSPZ) who underwent long-term CHMI (24 weeks after the last dose of PfSPZ). Panel G shows net optical density (OD) 1.0 anti-PfCSP antibodies by an enzyme-linked immunosorbent assay (ELISA) comparing vaccinated Tanzanian volunteers to volunteers in other trials receiving the same regimen. After five doses of 2.70 × 10⁵ PfSPZ/dose, volunteers in bagamoyo sporozoite vaccine 1 (BSPZV1) (N = 25) had a 4.3-fold lower median net OD 1.0 than those in the U.S.-based clinical trial Walter Reed Army Institute of Research (WRAIR) 2080 (N = 26) but a 6.6-fold higher median OD 1.0 than volunteers in 14-I-N010 in Bamako, Mali (N = 42), where malaria transmission rates are higher. There was a significant difference between the results for WRAIR 2080 vs. BSPZV1 (P = 0.0012), WRAIR 2080 vs. 14-I-N010 (P < 0.0001), and even 14-I-N010 vs. BSPZV1 (P = 0.002) (two-tailed t-test). AFU = arbitrary fluorescence units; aIFA = antibodies by immunofluorescence assay.

Figure 3.

Antibody responses to Plasmodium falciparum Sporozoites (PfSPZ) and PfCSP before controlled human malaria infection (CHMI). For all assays, uninfected subjects are shown as filled (black) circles and infected subjects are open circles. For each of the defined subject groups, the interquartile ranges and the median values of response of subjects in each group are shown. Assessment of antibodies was performed in sera from subjects before immunization and before CHMI #1 (∼2 weeks after the last dose of PfSPZ Vaccine or normal saline [NS]) and/or CHMI #2 (∼24 weeks after last dose of PfSPZ or NS) (A, D). Antibodies to PfCSP by ELISA are reported as net optical density (OD) 1.0 (the difference in OD 1.0 between pre-CHMI and preimmunization sera). (B, E) Antibodies to PfSPZ by aIFA are reported as net AFU 2 × 10⁵, the reciprocal serum dilution at which the fluorescent units were 2 × 10⁵ (AFU 2 × 10⁵) in pre-CHMI minus preimmunization sera. (C, F) Results of inhibition of sporozoite invasion (ISI) assay are reported as serum dilution at which there was 80% reduction of the number of PfSPZ that invaded a human hepatocyte line (HC-04) in the presence of pre-CHMI as compared with preimmunization sera from the same subject. Panels A–C show groups 2 (five doses of 1.35 × 10⁵ PfSPZ) and 3 (five doses of 2.7 × 10⁵ PfSPZ) before short-term CHMI (2 weeks after the last dose of PfSPZ or NS) and panels D–F show those volunteers in Groups 3 (five doses of 2.7 × 10⁵ PfSPZ) and 4 (five doses of 2.7 × 10⁵ PfSPZ) who underwent long-term CHMI (24 weeks after the last dose of PfSPZ). Panel G shows net optical density (OD) 1.0 anti-PfCSP antibodies by an enzyme-linked immunosorbent assay (ELISA) comparing vaccinated Tanzanian volunteers to volunteers in other trials receiving the same regimen. After five doses of 2.70 × 10⁵ PfSPZ/dose, volunteers in bagamoyo sporozoite vaccine 1 (BSPZV1) (N = 25) had a 4.3-fold lower median net OD 1.0 than those in the U.S.-based clinical trial Walter Reed Army Institute of Research (WRAIR) 2080 (N = 26) but a 6.6-fold higher median OD 1.0 than volunteers in 14-I-N010 in Bamako, Mali (N = 42), where malaria transmission rates are higher. There was a significant difference between the results for WRAIR 2080 vs. BSPZV1 (P = 0.0012), WRAIR 2080 vs. 14-I-N010 (P < 0.0001), and even 14-I-N010 vs. BSPZV1 (P = 0.002) (two-tailed t-test). AFU = arbitrary fluorescence units; aIFA = antibodies by immunofluorescence assay.

Figure 4.

Plasmodium falciparum Sporozoites (PfSPZ)–specific T-cell responses in vaccine recipients receiving 1.35 × 10⁵ PfSPZ. (A–D) PfSPZ-specific T-cell responses. Frequency of cytokine-producing memory CD4 T cells responding to (A) PfRBC or (B) PfSPZ. Throughout, “naïve T cell” refers to cells that co-express CCR7 and CD45RA, and “memory T cell” refers to all other T cells. Frequency of cytokine-producing memory CD8 T cells responding to (C) PfRBC or (D) PfSPZ. Results are the percentage of memory T cells producing interferon gamma, interleukin 2, and/or tumor necrosis factor alpha following stimulation minus the percentage of cells following control stimulation. (E) Frequency of the Vδ2⁺ subfamily of γδ T cells of total lymphocytes. Results are expressed as fold-change from the prevaccine frequency. (F) γδ T-cell activation in vivo. Data are the percentage of memory γδ T cells expressing HLA-DR and CD38 as measured on PBMCs following incubation with control stimulation (vaccine diluent). (G) Prevaccine frequency of PfRBC-specific Tregs in Tanzania compared with malaria-naïve U.S. subjects from the Vaccine Research Center (VRC) 314 study. (H) Frequency of PfRBC-specific Treg. Results are the percentage of CD4⁺Foxp3⁺CD25⁺CD127⁻ T cells expressing CD137 (also known as 4-1BB) after stimulation with Pf red blood cell (PfRBC) minus the percentage of cells following stimulation with uninfected RBC. (I) Percentage of total CD4 (left) or CD8 (right) T cells that are naïve (gray bar; CCR7⁺CD45RA⁺) or memory (blue bar; not CCR7⁺CD45RA⁺) phenotype assessed prevaccination in all 48 subjects vaccinated in Tanzania or in 14 healthy U.S. subjects from the VRC 314 study. For A–F and H, N = 24, and statistical difference was measured by using the Wilcoxon matched-pairs signed rank test. For G and I, statistical difference was measured by using the Mann–Whitney U test. P values are reported as not significant (ns), < 0.05 (*), < 0.01 (**), or < 0.001 (***). Data are mean ± SEM. Time points are prevaccine, 2 weeks after the first vaccination, and 2 weeks after the final vaccination. Black arrowhead designates PfSPZ Vaccine administration. This figure appears in color at www.ajtmh.org.