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.

Conflict of interest statement

The authors have read the journal's policy and have the following competing interests: TLR is a full time salaried employee of Sanaria Inc. This affiliation does not alter our adherence to PLOS One Policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Figures

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.