Background
Plasmodium vivax malaria is easily transmitted and remains dormant in the liver, with repeated relapses contributing to the clinical burden. Therefore, new treatments are needed to prevent P. vivax malaria. Targeted therapies that inhibit hepatocyte and erythrocyte invasion are crucial for reducing the overall disease burden and eliminating Plasmodium (Pv). A crucial component of the invasion mechanism used by sporozoites and merozoites is the interaction between apical membrane antigen 1 (AMA1) and the extracellular β-hairpin loop of the C-terminal portion of Okinawa neck protein 2 (RON2). Blocking the interaction of PfAMA1 or PkAMA1 with PfRON2 or PkRON2 using antibodies or peptides inhibits invasion, confirming that the AMA1-RON2 interaction is important for the Plasmodium life cycle. Utilizing human monoclonal antibodies against sporozoite antigens may be a promising approach for preventing malaria infection and related diseases.
On December 4, 2024, Nature Communications published an article titled "Potent AMA1-specific human monoclonal antibody against Plasmodium vivax Pre - erythrocytic and Blood Stages." The study identified 12 human monoclonal antibodies specific for Plasmodium vivax apical membrane antigen 1 (PvAMA1) from peripheral blood mononuclear cells of individuals exposed to Pv. These antibodies were shown to block PvAMA1 binding to PvRON2. PvAMA1 is required for sporozoite and merozoite invasion, making it a unique therapeutic target. The study found that the most potent humAb (designated 826827) blocked Pv infection in the chimeric FRG-humHep mouse model, preventing RON2 ring binding and consistently preventing blood-stage and sporozoite infection in vitro and in vivo against multiple clinical isolates.
Isolation and expression of monoclonal antibodies
Researchers isolated 12 human PvAMA1-specific monoclonal antibodies from the peripheral blood of Pv-exposed individuals. Clones for humAb generation were selected based on the presence of corresponding immunoglobulin light chains (IGLs), sequence quality, degree of somatic hypermutation (SHM), and selection of an IGH+IGL pair from B cells. The 12 antibodies were tested for binding to recombinant AMA1 in a multiplex immunoassay. Human Abs 806807, 826827, 832833, and 838839 exhibited the highest affinity. Notably, humAb 826827 exhibited higher affinity for PkAMA1 than for PvAMA1. The affinity of the humAbs for PvAMA1 ranged from 10.7×10⁻⁹ to 47.7× 10⁻⁹ M, as determined by SPR.
HumA b inhibits blood-stage infection
Using a modified Pf parasite line expressing PvAMA1 as a model, the ability of humAbs to inhibit merozoite invasion was evaluated. An IC50 curve analysis of four human monoclonal antibodies, 808809, 826827, 828829, and 830831, revealed that antibody 826827 exhibited the highest inhibition, with an IC50 of 2.6µg/ml.
Only humAb 826827 significantly inhibited Pv invasion of reticulocytes compared to the control humAb 043038. A dose-response invasion assay was performed against four additional Pv clinical isolates using humAb 826827 at concentrations ranging from 7.8 to 1000 µg/mL to determine its IC50. The average IC50 obtained for the four clinical isolates was 48 µg/mL (±6.6 SEM). These results, along with in vitro experiments with Pf-PvAMA1 transgenic parasites, demonstrate that human 826827 is highly effective in inhibiting AMA1-dependent erythrocyte invasion.
Inhibitory effect of HumAb on Pv sporozoites in human hepatocytes
HumaB 826827 has been shown to inhibit sporozoite invasion of human hepatocytes in vitro with an IC50 value of 3.7 µg/mL.
Using a liver chimeric mouse model (FRG-humHep) demonstrated that mice administered 300 μg of humAb 826827 had significantly fewer liver-stage parasites compared to a control humAb, as assessed by 18S quantitative RT-qPCR. HumAb 826827 significantly reduced sporozoite invasion of hepatocytes in vivo.
Structural studies of the interaction between humAb 826827 and PvAMA1
humAb 826827 primarily interacts with the mobile loop portion of Domain 1 and Domain 2 of PvAMA1, regions critical for PvAMA1 function. Five of the six complementarity-determining regions (CDRs) (i.e., L1, L2, H1, H2, and H3) form direct contacts with PvAMA1 , with the CDR-H3 loop contributing 70% of the buried surface area. The CDR-H3 loop forms a disulfide-bridged β-hairpin structure that occupies the hydrophobic groove of PvAMA1 Domain 1, the binding site for the RON2 loop peptide . humAb 826827 interacts with the PvAMA1 Domain 2 loop through CDR-H3 and CDR-H1, forming a salt bridge and multiple hydrogen bonds, stabilizing the loop's position within Domain 1. Binding of humAb 826827 prevents the displacement of the Domain 2 loop, which is required for the interaction of RON2 with AMA1, and thus humAb 826827 effectively blocks this critical parasite invasion step.
The contact residues between PvAMA1 and the RON2 loop overlap with those of PvAMA1 that contact the CDR-H3 residues of humAb 826827. Sequence and structural analysis of the contact residues showed that the epitope of humAb 826827 is highly conserved with PkAMA1 and PcAMA1.
humAb 826827 competes with PvRON2 for the same epitope on PvAMA1, confirming its inhibitory mechanism. Despite the polymorphism of PvAMA1, the epitope of humAb 826827 is highly conserved across multiple clinical isolates, indicating that humAb 826827 has broad inhibitory potential against different Pv strains.
Summarize
This study identified a highly conserved PvAMA1 epitope that can be targeted by humAb 826827, preventing AMA1-dependent sporozoite invasion of hepatocytes and schistospore invasion of reticulocytes in vitro and in vivo. HumAb 826827 may lead to the development of new therapeutics to combat Pv infection, disease, and transmission. Furthermore, the identification of a conserved inhibitory epitope on PvAMA1 that is effectively targeted by HumAb 826827 may guide structure-based Pv vaccine design. Future studies in nonhuman primate models may provide valuable data supporting the development of humAb 826827 as a therapeutic or prophylactic agent.
