IMRAS-A clinical trial of mosquito-bite immunization with live, radiation-attenuated P. falciparum sporozoites: Impact of immunization parameters on protective efficacy and generation of a repository of immunologic reagents

Abstract

Background: Immunization with radiation-attenuated sporozoites (RAS) by mosquito bite provides >90% sterile protection against Plasmodium falciparum (Pf) malaria in humans. RAS invade hepatocytes but do not replicate. CD8+ T cells recognizing parasite-derived peptides on the surface of infected hepatocytes are likely the primary protective mechanism. We conducted a randomized clinical trial of RAS immunization to assess safety, to achieve 50% vaccine efficacy (VE) against controlled human malaria infection (CHMI), and to generate reagents from protected and non-protected subjects for future identification of protective immune mechanisms and antigens.

Methods: Two cohorts (Cohort 1 and Cohort 2) of healthy, malaria-naïve, non-pregnant adults age 18-50 received five monthly immunizations with infected (true-immunized, n = 21) or non-infected (mock-immunized, n = 5) mosquito bites and underwent homologous CHMI at 3 weeks. Immunization parameters were selected for 50% protection based on prior clinical data. Leukapheresis was done to collect plasma and peripheral blood mononuclear cells.

Results: Adverse event rates were similar in true- and mock-immunized subjects. Two true- and two mock-immunized subjects developed large local reactions likely caused by mosquito salivary gland antigens. In Cohort 1, 11 subjects received 810-1235 infected bites; 6/11 (55%) were protected against CHMI vs. 0/3 mock-immunized and 0/6 infectivity controls (VE 55%). In Cohort 2, 10 subjects received 839-1131 infected bites with a higher first dose and a reduced fifth dose; 9/10 (90%) were protected vs. 0/2 mock-immunized and 0/6 controls (VE 90%). Three/3 (100%) protected subjects administered three booster immunizations were protected against repeat CHMI vs. 0/6 controls (VE 100%). Cohort 2 uniquely showed a significant rise in IFN-γ responses after the third and fifth immunizations and higher antibody responses to CSP.

Conclusions: PfRAS were generally safe and well tolerated. Cohort 2 had a higher first dose, reduced final dose, higher antibody responses to CSP and significant rise of IFN-γ responses after the third and fifth immunizations. Whether any of these factors contributed to increased protection in Cohort 2 requires further investigation. A cryobank of sera and cells from protected and non-protected individuals was generated for future immunological studies and antigen discovery.

Trial registration: ClinicalTrials.gov NCT01994525.

Fig 1. Cohort 1: Flow diagram of immunized and control subjects.

Twenty-four subjects met all eligibility criteria and 14 were assigned to the true-immunization group, four were assigned to the mock-immunization group and the remaining six subjects were assigned as infectivity controls.

Fig 2. Cohort 2: Flow diagram of immunized and control subjects.

Twenty-two subjects met all eligibility criteria and 12 were assigned to the true-immunization group, four were assigned to the mock-immunization group and the remaining six subjects were assigned as infectivity controls.

Fig 3. Cumulative numbers of bites in Cohort 1 and Cohort 2.

The cumulative numbers of P. falciparum RAS-infected mosquitoes administered at immunizations 1–5 for each protected and non-protected subjects and the average cumulative total for all subjects. Panel A: Cohort 1. Panel B: Cohort 2.

Fig 4. Development of parasitemia in the true- and mock-immunized and infectivity control subjects.

Parasitemia-free survival curves (Kaplan-Meier) for true- and mock-immunized subjects and infectivity controls based on microscopic examination of peripheral blood smears. Panel A: Cohort 1 where 11 true-immunized, 3 mock-immunized and 6 infectivity controls received CHMI. Panel B: Cohort 1 where 10 true-immunized, 2 mock-immunized and 6 infectivity controls received CHMI.

Fig 5. Comparison of total number of infectious bites and time to CHMI in Cohort 1 and Cohort 2 with the target range of infectious bites.

The total number of infectious bites and days between final immunization and CHMI are compared for Cohort 1 (protected: blue circles; non-protected: open circles) and Cohort 2 (protected: green triangles; non-protected: open triangles). All Cohort 1 and Cohort 2 subjects fell within the target box except one protected subject and one non-protected subject in Cohort 1 that fell just outside the target box. The distribution of subjects in Cohort 1 and Cohort 2 showed no association of protection with total numbers of infectious bites or time (days) before CHMI.

Fig 6. Cohort 1 and Cohort 2: FluoroSpot activities to whole sporozoites after each immunization and CHMI.

FluoroSpot IFN-γ, IL2 and IFN-γ+IL2 activities to whole sporozoites were measured pre-immunization (Pre), after each immunization (1, 2, 3, 4, 5) and after CHMI (Post-C); subjects in Cohort 1 only were also measured 7 days after the first immunization (1*). Cohort 1 and Cohort 2 protected and non-protected subjects are shown by indicated symbols. Geometric means are indicated by red bars. Panel A: IFN-γ: Geometric mean activities in both Cohorts significantly rose (p = <0.001) after the first immunization and were higher in Cohort 1 than Cohort 2 after the first and second immunizations (see text). In Cohort 2, activities significantly rose after the fifth immunization. Panel B: IL2: Geometric mean activities in both Cohorts significantly rose (p = <10⁻³) after the first and second (p = <0.01) immunizations and activities of Cohort 1 were significantly higher than Cohort 2 after the first and second immunizations (see text). In Cohort 2, activities significantly rose after the third and fifth immunizations. Panel C: IFN-γ+IL2: Geometric mean activities in both Cohorts 1 were significantly higher after the first immunization, and activities of Cohort 1 were higher than Cohort 2 after the first and second immunizations (see text). In Cohort 2, activities significantly rose after the third and fifth immunizations.

Fig 7. Cohort 1 and Cohort 2: ELISA activities after third and fifth immunizations and CHMI, and IFA activities after fifth immunization and CHMI.

ELISA activities to CSP repeat, CSP full length (FL) and AMA1 of Cohort 1 (blue circles) and Cohort 2 (green triangles) were measured pre-immunization (Pre), 14 days after the third (3) and 22 days after fifth (5) immunizations and 28 days after CHMI (Post-CHMI). IFA activities to sporozoites were measured pre-immunization (Pre), and 22 days after fifth (5) immunizations / pre-CHMI. Protected subjects (closed symbols) and non-protected subjects (open symbols). The Geometric mean (red bar) is shown for all subjects in each Cohort. Panel A: CSP repeat: Geometric mean activities in both Cohorts significantly rose after the third and fifth immunizations (see text). Activities in Cohort 2 were significantly higher than Cohort 1 after the third and fifth immunizations. Panel B: CSP Full Length (FL): activities were similar to those with CSP repeat and activities of Cohort 2 were only significantly higher than Cohort 1 after the third and fifth immunizations. Panel C: AMA1: Geometric mean activities of Cohort 1 and Cohort 2 significantly rose after the third and fifth immunization (see text) but were not different between Cohort 1 and Cohort 2. Panel D IFA: Activities of Cohort 1 and Cohort 2 significantly rose after the fifth immunizations (see text) but were similar in Cohort 1 and Cohort 2.