Protective Efficacy of Plasmodium vivax Radiation-Attenuated Sporozoites in Colombian Volunteers: A Randomized Controlled Trial

Abstract

Background: Immunizing human volunteers by mosquito bite with radiation-attenuated Plasmodium falciparum sporozoites (RAS) results in high-level protection against infection. Only two volunteers have been similarly immunized with P. vivax (Pv) RAS, and both were protected. A phase 2 controlled clinical trial was conducted to assess the safety and protective efficacy of PvRAS immunization.

Methodology/principal findings: A randomized, single-blinded trial was conducted. Duffy positive (Fy+; Pv susceptible) individuals were enrolled: 14 received bites from irradiated (150 ± 10 cGy) Pv-infected Anopheles mosquitoes (RAS) and 7 from non-irradiated non-infected mosquitoes (Ctl). An additional group of seven Fy- (Pv refractory) volunteers was immunized with bites from non-irradiated Pv-infected mosquitoes. A total of seven immunizations were carried out at mean intervals of nine weeks. Eight weeks after last immunization, a controlled human malaria infection (CHMI) with non-irradiated Pv-infected mosquitoes was performed. Nineteen volunteers completed seven immunizations (12 RAS, 2 Ctl, and 5 Fy-) and received a CHMI. Five of 12 (42%) RAS volunteers were protected (receiving a median of 434 infective bites) compared with 0/2 Ctl. None of the Fy- volunteers developed infection by the seventh immunization or after CHMI. All non-protected volunteers developed symptoms 8-13 days after CHMI with a mean pre-patent period of 12.8 days. No serious adverse events related to the immunizations were observed. Specific IgG1 anti-PvCS response was associated with protection.

Conclusion: Immunization with PvRAS was safe, immunogenic, and induced sterile immunity in 42% of the Fy+ volunteers. Moreover, Fy- volunteers were refractory to Pv malaria.

Trial registration: Identifier: NCT01082341.

Fig 1. Trial flow diagram.

Number of individuals in the screening, immunization, and CHMI steps.

Fig 2. Study design.

Immunization schedule for the three groups of volunteers (RAS, Ctl, and Fy-) who received seven immunizations and then were challenged with P. vivax field isolate infected mosquitoes.

Fig 3. Parasitemia determined by qPCR.

A. Number of parasite DNA copies per μL determined during the immunization phase in the Fy- group. B. Parasitemia after the CHMI in Ctl and RAS groups. Ctl* corresponds to the parasitemia dynamics determined by qPCR during a previous CHMI experiment that included naïve Fy+ individuals using the same procedures for comparison. Each point represents mean ± SEM of parasites/μL (Log₁₀).

Fig 4. Frequency and intensity of adverse events after the CHMI.

The adverse events graded according to FDA recommendations [22] and grouped as fever-related symptoms; gastric symptoms; and, others in RAS (n = 12; A) and Ctl group (n = 2; B) are shown. No AE after the CHMI were observed in the Fy- group (n = 5). Abbreviations: Abd, abdominal pain; rash, generalized rash; aphthous, aphthous stomatitis.

Fig 5. Antibody response against PvCS-NRC peptide.

Total IgG response determined by ELISA in the RAS group (n = 12; A), Fy- group (n = 5; B) and Ctl group (n = 2; red line in A and B). Values are expressed as reactivity index (RI) defined as sample OD at 1:200 serum dilutions divided by the cut-off value. Mean ± SEM are shown. C. Correlations between total received dose of infective bites and RI at seventh immunization for RAS and Fy- volunteers. Spearman’s rank correlation (r_s) and p values are shown. D. Mean ± SEM of RI for protected and non-protected volunteers after every immunization. * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig 6. IgG isotype response against PvCS-NRC peptide.

Antibody IgG isotype levels determined by ELISA in the RAS group (n = 12; A), Fy- group (n = 5; B) and Ctl group (n = 2; B) at seventh immunization are shown. Values are expressed as reactivity index (RI) defined as sample OD divided by the cut-off value. Horizontal bars indicate median values. p value using the Mann-Whitney U test between protected and non-protected are shown.

Fig 7. IFN-γ production to individual malaria antigens before the CHMI.

Ex-vivo IFN-γ ELISpot responses in the RAS group (n = 12; A), Fy- group (n = 5; B), and Ctl group (n = 2; red line in A and B) previous to the CHMI. PBMC were stimulated with Pv spz lysate, PvCS-NRC, and PvTRAP. Mean ± SEM are shown. C. Mean ± SEM of spots per 10⁶ PBMC for protected and non-protected volunteers.