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. 2023 Jul 15;22(1):210.
doi: 10.1186/s12936-023-04638-8.

The malaria blood stage antigen PfCyRPA formulated with the TLR-4 agonist adjuvant GLA-SE elicits parasite growth inhibitory antibodies in experimental animals

Marco Tamborrini  1   2 , Anja Schäfer  1   2 , Julia Hauser  1   2 , Linghui Zou  1   2 , Daniel H Paris  1   2 , Gerd Pluschke  3   4
Affiliations

The malaria blood stage antigen PfCyRPA formulated with the TLR-4 agonist adjuvant GLA-SE elicits parasite growth inhibitory antibodies in experimental animals

Marco Tamborrini et al. Malar J. .

Abstract

Background: Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. In contrast to several previously clinically tested merozoite vaccine candidate antigens, PfCyRPA is not polymorphic, making it a promising candidate antigen for blood stage vaccine development.

Methods: Mice and rabbits were immunized with vaccine formulations of recombinantly expressed PfCyRPA adjuvanted either with the glucopyranosyl lipid A (GLA) containing adjuvants GLA-LSQ, GLA-SE, GLA-Alum or with Nanoalum. ELISA and indirect immunofluorescence assays (IFA) were used to analyse elicited IgG titers and the P. falciparum growth inhibitory activity was determined with a standardized in vitro [3H]-hypoxanthine incorporation assay.

Results: In the mouse experiments, the GLA adjuvanted formulations were superior to the Nanoalum formulation with respect to antibody titer development, IFA sero-conversion rates and in vitro parasite growth-inhibitory activity. In rabbits, the highest titers of parasite growth inhibitory antibodies were obtained with the GLA-SE formulation. Comparable mean ELISA IgG endpoint titers were reached in rabbits after three immunizations with GLA-SE adjuvanted PfCyRPA doses of 5, 25 and 100 µg, but with 100 µg of antigen, only two immunizations were required to reach this titer.

Conclusion: PfCyRPA formulated with the human-compatible adjuvant GLA-SE represents an attractive vaccine candidate for early clinical testing in a controlled P. falciparum blood stage challenge trial.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
PfCyRPA adjuvanted with either GLA-LSQ, GLA-SE, GLA-Alum or Nanoalum elicited in mice high IgG titers with in vitro parasite growth-inhibitory activity. ELISA readouts (optical density [OD] means ± standard deviations) obtained with serial dilution of mouse sera taken pre-immune and after each immunization are shown in a–e. Serum anti-PfCyRPA IgG ELISA endpoint titers of individual animals are shown in f–j and lines represent the mean titer. Determinations of IgG subclass profiles by ELISA using plates coated with recombinant PfCyRPA are shown in k–o. Sera from mice collected after the third immunization were tested individually at a serum dilution of 1:6400. Shown are OD values obtained with individual sera and means (colored bars). In vitro parasite growth-inhibitory activities of purified total IgG from pooled sera of each group of mice are shown in p–t. Dots represent duplicate replicates of two independent [3H]-hypoxanthine incorporation assays. For each group a four-parameter sigmoidal dose-response curve was fitted to the relationship between the log10 of the IgG concentration and percentage inhibition, and then used to interpolate IC50 values
Fig. 2
Fig. 2
PfCyRPA adjuvanted with either GLA-LSQ, GLA-SE or GLA-Alum elicited in rabbits high IgG titers with in vitro parasite growth-inhibitory activity. ELISA readouts (means ± standard deviations) obtained with serial dilution of sera taken pre-immune and after each immunization are shown in a–d. Serum anti-PfCyRPA IgG ELISA endpoint titers of individual animals are shown in e–h, with lines representing the mean titer. In vitro parasite growth-inhibitory activities of purified total IgG from individual rabbit serum samples collected after the final immunization are shown in i–l. Dots represent duplicate replicates of two independent [3H]-hypoxanthine incorporation assays. For each individual rabbit IgG preparation, a four-parameter sigmoidal dose-response curve was fitted to the relationship between log10 of the antibody concentration and percentage inhibition, and then used to interpolate IC50 values. Purified IgG from non-immunized animals and the parasite inhibitory anti-PfCyRPA mAb c12 were used as controls (m). The mean ELISA IgG endpoint titers obtained after the second, but not after the third immunization were significantly higher in the adjuvanted immunization groups when compared to the unadjuvanted group (Additional file 1: Table S1)
Fig. 3
Fig. 3
Dose‑response analysis in rabbits. Groups of rabbits (n = 3) were immunized three times with three different doses of PfCyRPA (5, 25 and 100 µg) adjuvanted with GLA-SE. Control animals were immunized with GLA-SE alone. ELISA readouts (means ± standard deviations) obtained with serial dilutions of sera taken pre-immune and after each immunization are shown in a–d. Serum anti-PfCyRPA IgG ELISA endpoint titers of individual animals are shown in e–h and lines represent the mean titer
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