Immunization with PfGBP130 generates antibodies that inhibit RBC invasion by P. falciparum parasites

Abstract

Background: Despite decades of effort, Plasmodium falciparum malaria remains a leading killer of children. The absence of a highly effective vaccine and the emergence of parasites resistant to both diagnosis as well as treatment hamper effective public health interventions.

Methods and results: To discover new vaccine candidates, we used our whole proteome differential screening method and identified PfGBP130 as a parasite protein uniquely recognized by antibodies from children who had developed resistance to P. falciparum infection but not from those who remained susceptible. We formulated PfGBP130 as lipid encapsulated mRNA, DNA plasmid, and recombinant protein-based immunogens and evaluated the efficacy of murine polyclonal anti-PfGBP130 antisera to inhibit parasite growth in vitro. Immunization of mice with PfGBP130-A (aa 111-374), the region identified in our differential screen, formulated as a DNA plasmid or lipid encapsulated mRNA, but not as a recombinant protein, induced antibodies that inhibited RBC invasion in vitro. mRNA encoding the full ectodomain of PfGBP130 (aa 89-824) also generated parasite growth-inhibitory antibodies.

Conclusion: We are currently advancing PfGBP130-A formulated as a lipid-encapsulated mRNA for efficacy evaluation in non-human primates.

Keywords: Plasmodium falciparum; blood stage malaria antigen; growth inhibiting activity; mRNA; malaria; vaccine.

Figure 1

Anti-PfGBP130-A generated by DNA, but not recombinant protein immunization markedly attenuates parasite replication in multiple parasite strains. (A) Polyclonal anti-PfGBP130-A antibodies generated by DNA immunization in mice inhibit parasite growth by 79-89% in multiple parasite strains in vitro. Ring stage parasites at 0.3% parsitemia were cultured in the presence of anti-PfGBP130 mouse sera at 1:10 dilution. Negative controls included no anti-sera and normal mouse sera. (B) Parasite growth assay performed as in (A), but with dilution series of anti-PfGBP130-A generated by DNA immunization. IC₅₀ for inhibition of parasite growth was 0.6% serum. (C) Polyclonal anti-PfGBP130-A antibodies generated by recombinant protein immunization in mice resulted in no or modest (22-29%) growth inhibition in multiple parasite strains in vitro. For (A, C), bars represent means, circles represent values from replicate wells, and error bars represent SEM. For (B), circles represent means, error bars represent SEM. Results in (A, C) representative of 5 independent experiments.

Figure 2

Domain structure of PfGBP130. PfGBP130 is an invariant, PEXEL containing merozoite surface antigen comprised of an N-terminal charged 225 aa domain followed by twelve copies of a 50 aa repeat domain. The PfGBP130 clone identified by differential screening (PfGBP130-A) encoded aa 111-374 which comprises the majority of the N terminal charged domain and three copies of the 50 aa repeat region.

Figure 3

PfGBP130-A formulated as a DNA plasmid or recombinant protein is immunogenic in mice. (A) Mice immunized with a DNA plasmid encoding PfGBP130-A generated low titer (1:8,000) antibodies against PfGBP130-A and PfGBP130-ecto coated beads with no reactivity against negative control protein (PfGARP-ecto) coated beads. (B) Mice immunized with recombinant protein encoding PfGBP130-A generated high titer (1:512,000) antibodies against PfGBP130-A and PfGBP130-ecto coated beads with negligible binding to a negative control protein (PfGARP-ecto) coated beads.

Figure 4

Antibodies to PfGBP130-A inhibit merozoite invasion. Polyclonal anti-PfGBP130-A antibodies generated by DNA immunization in mice inhibit merozoite invasion by 88% in vitro. Schizont stage parasites were cultured in the presence of anti-PfGBP130-A mouse sera at 1:10 dilution for 12 hours and newly invaded ring-stage parasites were enumerated. Negative controls included media alone and normal mouse sera. Bars represent means, circles represent values from replicate wells, and error bars represent SEM.

Figure 5

Immunization with PfGBP130-A or PfGBP130-ecto formulated as lipid encapsulated mRNA generates antibodies that markedly attenuate parasite replication. Immunization with (A) lipid encapsulated mRNA encoding PfGBP130-A or (B) PfGBP130-ecto generates high titer (both 1:512,000), specific antibodies against PfGBP130-A and PfGBP130-ecto coated beads with no reactivity against negative control protein (PfGARP-ecto) coated beads. (C) Polyclonal murine anti-PfGBP130 antibodies generated by immunization with LNPs containing mRNA encoding PfGBP130-A or PfGBP130-ecto inhibit parasite growth by 80% in vitro. Ring stage parasites were cultured in the presence of anti-PfGBP130 mouse sera at 1:10 dilution. Negative controls included no anti-sera and normal mouse sera. For (A, B) circles represent means, error bars represent SEM. For (C), bars represent means, circles represent values from replicate wells, and error bars represent SEM. Results in (C) representative of 3 independent experiments.