Single immunization with genetically attenuated Pf∆mei2 (GA2) parasites by mosquito bite in controlled human malaria infection: a placebo-controlled randomized trial

Abstract

Malaria vaccines consisting of metabolically active Plasmodium falciparum (Pf) sporozoites can offer improved protection compared with currently deployed subunit vaccines. In a previous study, we demonstrated the superior protective efficacy of a three-dose regimen of late-arresting genetically attenuated parasites administered by mosquito bite (GA2-MB) compared with early-arresting counterparts (GA1-MB) against a homologous controlled human malaria infection. Encouraged by these results, we explored the potency of a single GA2-MB immunization in a placebo-controlled randomized trial. Primary outcomes were safety and tolerability, time-to-parasitemia and protective efficacy. Humoral and cellular immunological results were considered secondary outcomes. Here we report the safe administration of GA2-MB with no breakthrough malaria and sterile protection in nine of ten participants at 6 weeks after a single immunization with 50 GA2-infected mosquitoes, compared with none of five mock-immunized participants, against a homologous controlled human malaria infection. Immunization increased circulating Pf-specific polyfunctional effector memory CD4⁺ T cells coexpressing tumor necrosis factor and interleukin-2. This unprecedented 90% protective efficacy after a single low-dose immunization holds great promise for the potency of GA2 immunization. Future studies should demonstrate whether GA2 is similarly efficacious in pre-exposed populations and whether the favorable safety profile reported here holds up in larger groups. ClinicalTrials.gov registration: NCT05468606 .

Fig. 1. CONSORT diagram on recruitment and inclusions.

Between 13 February 2023 and 17 March 2023, 27 persons were screened, of whom 15 were included, randomized, (mock-)immunized and infected with wild-type malaria parasites in a CHMI.

Fig. 2. Study design, immunization dose and protection against CHMI.

a, Schematic overview of study design. b, Number of blood-fed mosquitoes per participant at immunization. Black horizontal lines represent the median. The dashed horizontal line represents the target dose (45–55 blood-fed mosquitoes). c, Kaplan–Meier curve of percentage of participants that had a negative PfqPCR (<100 parasites per ml) in peripheral blood after CHMI. X, censored; log-rank test, P < 0.0001.

Fig. 3. Prominent polyfunctional CD4⁺ memory T cell response in GA2 single-immunized participants.

a, Plasma antibody levels against the indicated Pf antigens on the day before CHMI (C−1). Values are log₁₀ transformed. b, Frequency of CD4⁺ and Vδ2⁺ γδ T cells expressing the indicated cytokines upon stimulation with Pf RBC corrected for unRBC stimulation. c, Frequency of CD4⁺ and Vδ2⁺ γδ T cells expressing single or more than one of the indicated cytokines per cell upon Pf RBC and unRBC stimulation. d, Frequency of CD45RA⁻ CD4⁺ (upper) and Vδ2⁺ γδ (lower) memory T cells (T_MEM) among polyfunctional cells at the indicated time points upon Pf RBC stimulation. e, Frequency of central (T_CM) (upper) and effector (T_EM) (lower) memory cells among polyfunctional CD4⁺ T cells. Filled circles and triangles indicate the data from individual participants and the horizontal black line indicates the arithmetic mean (a, d, e). Filled circles and error bars indicate the arithmetic mean and s.e.m., respectively (b). Bar charts represent arithmetic means and error bars represent s.e.m. (c). Two-tailed Mann–Whitney test (a, b (at C−1), d, e). PfAMA-1, Pf apical membrane antigen-1 ; PfCSP, Pf circumsporozoite protein; Pf MSP-1, Pf merozoite surface protein-1.

Extended Data Fig. 1. CHMI dose and time-to-parasitaemia.

A. Number of infected blood fed mosquitoes per participant at the controlled human malaria infection (CHMI). A black horizontal line represents the median. B. Development of parasitaemia per day after CHMI. Lines with different symbols represent different participants. The horizontal dotted grey line represents the cut-off for parasitaemia (100 parasites/mL blood) at which treatment is given. The horizontal continuous grey line represents the lowest limit of detection of the assay (50 parasites/mL blood).

Extended Data Fig. 2. Representative gating strategy to define T cell subsets.

Live single cells were defined as NK cells (CD3⁻CD56⁺), CD4 or CD8 effector (CCR7⁻CD45RA⁻) and central (CCR7⁺CD45RA⁻) memory T cells and Vδ2⁺ or Vδ2⁻ γδ T cells, based on the surface marker expression.

Extended Data Fig. 3. Representative cytokine expression gating strategy.

Gates defining the indicated cytokine expressing CD4⁺ (A), CD8⁺ (B), NK (C), Vδ2⁺ γδ (D) and Vδ2⁻ γδ (E) T cells upon stimulation with Pf infected (Pf RBC; top) and uninfected RBC (unRBC).

Extended Data Fig. 4. Cytokine expression in T cell subsets.

CD8⁺ (A), Vδ2^- γδ (B) and, NK (C) T cells expressing the indicated cytokines upon stimulation with Pf infected RBC (Pf RBC) reference corrected for uninfected RBC (unRBC) stimulation. Data corresponding to GA2-MB and placebo groups are indicated in orange and purple, respectively (A–C). Filled circles and error bars indicate arithmetic mean and standard error mean, respectively. Two tailed Mann-Whitney test (at C-1).

Extended Data Fig. 5. Mono and polyfunctional T cells moderately co-expressing Th2 cytokines.

Frequency of CD4⁺ (A) and Vδ2⁺ γδ (B) T cells expressing the indicated cytokines in combination with or without type-2 cytokines (Th2: IL-4, IL-5 and IL-13) among GA2-MB and mock-immunised participants upon Pf RBC (left) and unRBC (right) stimulation. Filled circles and error bars indicate arithmetic mean and standard error of mean of both intervention groups, respectively.

Extended Data Fig. 6. Memory status of T cell subsets.

A,B. CD45RA expression in CD4⁺ (A) and Vδ2⁺ γδ (B) T cells among cytokine expressing cells at baseline (I-1) and C-1 upon Pf RBC stimulation. C-F. Frequency of CD45RA⁻ mono^- (C,E) and polyfunctional (D,F) CD4⁺ (C,D) and Vδ2⁺ γδ (E,F) memory T cells (T_MEM) upon Pf RBC and unRBC stimulation. G,H. Frequency of central (T_CM; top) and effector (T_EM; bottom) memory cells among monofunctional and polyfunctional in CD4⁺ (G) and Vδ2⁺ γδ (H) T cells. Each data point represent individual participant and data corresponding to GA2-MB and placebo groups are indicated in orange and purple, respectively (C-H). Horizontal lines indicate arithmetic mean (C-H). Two tailed Mann-Whitney test.