RESUMEN
New therapeutics are a priority for preventing and eliminating Plasmodium vivax (Pv) malaria because of its easy transmissibility and dormant stages in the liver. Relapses due to the dormant liver stages are the major contributor to reoccurring Pv. Therefore, therapies that reduce the establishment of dormant parasites and blood-stage infection are important for controlling this geographically widespread parasite. Here, we isolated 12 human monoclonal antibodies (humAbs) from the plasma of a Pv-exposed individual that recognized Pv apical membrane antigen 1 (PvAMA1). PvAMA1 is important for both sporozoite invasion of hepatocytes and merozoite invasion of reticulocytes. We identified one humAb, 826827, that blocked invasion of human erythrocytes using a transgenic P. falciparum line expressing PvAMA1 (IC 50 = 3 µg/mL) and all Pv clinical isolates in vitro . This humAb also inhibited sporozoite invasion of a human hepatocyte cell line and primary human hepatocytes (IC 50 of 0.3 - 3.7 µg/mL). The crystal structure of recombinant PvAMA1 with the antigen-binding fragment of 826827 at 2.4 Å resolution shows that the humAb partially occupies the highly conserved hydrophobic groove in PvAMA1 that binds its known receptor, RON2. HumAb 826827 binds to PvAMA1 with higher affinity than RON2, accounting for its potency. To our knowledge, this is the first reported humAb specific to PvAMA1, and the PvAMA1 residues it binds to are highly conserved across different isolates, explaining its strain-transcendent properties.
RESUMEN
BACKGROUND: Plasmodium vivax has been more resistant to various control measures than Plasmodium falciparum malaria because of its greater transmissibility and ability to produce latent parasite forms. Therefore, developing P. vivax vaccines and therapeutic monoclonal antibodies (humAbs) remains a high priority. The Duffy antigen receptor for chemokines (DARC) expressed on erythrocytes is central to P. vivax invasion of reticulocytes. P. vivax expresses a Duffy binding protein (PvDBP) on merozoites, a DARC ligand, and the DARC: PvDBP interaction is critical for P. vivax blood stage malaria. Therefore, PvDBP is a leading vaccine candidate for P. vivax and a target for therapeutic human monoclonal antibodies (humAbs). METHODS: Here, the functional activity of humAbs derived from naturally exposed and vaccinated individuals are compared for the first time using easily cultured Plasmodium knowlesi (P. knowlesi) that had been genetically modified to replace its endogenous PkDBP orthologue with PvDBP to create a transgenic parasite, PkPvDBPOR. This transgenic parasite requires DARC to invade human erythrocytes but is not reticulocyte restricted. This model was used to evaluate the invasion inhibition potential of 12 humAbs (9 naturally acquired; 3 vaccine-induced) targeting PvDBP individually and in combinations using growth inhibition assays (GIAs). RESULTS: The PvDBP-specific humAbs demonstrated 70-100% inhibition of PkPvDBPOR invasion with the IC50 values ranging from 51 to 338 µg/mL for the 9 naturally acquired (NA) humAbs and 33 to 99 µg/ml for the 3 vaccine-induced (VI) humAbs. To evaluate antagonistic, additive, or synergistic effects, six pairwise combinations were performed using select humAbs. Of these combinations tested, one NA/NA (099100/094083) combination demonstrated relatively strong additive inhibition between 10 and 100 µg/mL; all combinations of NA and VI humAbs showed additive inhibition at concentrations below 25 µg/mL and antagonism at higher concentrations. None of the humAb combinations showed synergy. Invasion inhibition efficacy by some mAbs shown with PkPvDBPOR was closely replicated using P. vivax clinical isolates. CONCLUSION: The PkPvDBPOR transgenic model is a robust surrogate of P. vivax to assess invasion and growth inhibition of human monoclonal Abs recognizing PvDBP individually and in combination. There was no synergistic interaction for growth inhibition with the humAbs tested here that target different epitopes or subdomains of PvDBP, suggesting little benefit in clinical trials using combinations of these humAbs.
