Mimotopes of Apical Membrane Antigen 1: Structures of Phage-Derived Peptides Recognized by the Inhibitory Monoclonal Antibody 4G2dc1 and Design of a More Active Analogue

Abstract

Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum is an integral membrane protein that plays a key role in merozoite invasion of host erythrocytes. A monoclonal antibody, 4G2dc1, recognizes correctly folded AMA1 and blocks merozoite invasion. Phage display was used to identify peptides that bind to 4G2dc1 and mimic an important epitope of AMA1. Three of the highest-affinity binders-J1, J3, and J7-were chosen for antigenicity and immunogenicity studies. J1 and J7 were found to be true antigen mimics since both peptides generated inhibitory antibodies in rabbits (J. L. Casey et al., Infect. Immun. 72:1126-1134, 2004). In the present study, the solution structures of all three mimotopes were investigated by nuclear magnetic resonance spectroscopy. J1 adopted a well-defined region of structure, which can be attributed in part to the interactions of Trp11 with surrounding residues. In contrast, J3 and J7 did not adopt an ordered conformation over the majority of residues, although they share a region of local structure across their consensus sequence. Since J1 was the most structured of the peptides, it provided a template for the design of a constrained analogue, J1cc, which shares a structure similar to that of J1 and has a disulfide-stabilized conformation around the Trp11 region. J1cc binds with greater affinity to 4G2dc1 than does J1. These peptide structures provide the foundation for a better understanding of the complex conformational nature of inhibitory epitopes on AMA1. With its greater conformational stability and higher affinity for AMA1, J1cc may be a better in vitro correlate of immunity than the peptides identified by phage display.Malaria infects 300 to 500 million people per year worldwide and causes 2 to 3 million deaths, mainly in children under 5 years of age. A considerable effort is being devoted to the development of a vaccine against malaria, and one of the leading candidates for inclusion in such a vaccine is apical membrane antigen 1 (AMA1), a type I integral membrane protein conserved throughout all Plasmodium species. AMA1 is critical for the invasion of host erythrocytes (36) and is translocated from the micronemes onto the parasite surface around the time of invasion (20). Although its precise role in invasion remains undefined, it has been postulated that AMA1 is involved in realignment of the parasite after attachment to the erythrocyte, ensuring that the apical prominence of the merozoite is in close proximity to the erythrocyte surface (8, 33). Recombinant AMA1 induced protective immune responses in mouse and monkey models of malaria (2, 10, 11, 13), and both monoclonal and polyclonal antibodies to AMA1 inhibit merozoite invasion of erythrocytes (2,9,11,12,25,34,42). The observation that it was not possible to obtain targeted gene disruptions of the AMA1 gene that knocked out the function of the protein further supports an important role for AMA1 in the invasion of host erythrocytes (43). Recently, a conditional knockout of Toxoplasma gondii AMA1 was cre...

Identification of an Immunogenic Broadly Inhibitory Surface Epitope of the Plasmodium vivax Duffy Binding Protein Ligand Domain

“…Phage display has been a useful tool for epitope mapping with the advantages of rapidly producing refolded protein, fused to phage coat, forming a traceable link back to the genotype (46, 47), and has been used to map epitopes on P. falciparum apical membrane antigen 1 (AMA1) (48–51), merozoite surface protein (MSP) (52), and circumsporozoite protein (CSP) (53–55). Additional immunogenicity studies demonstrated the ability of the 3C9 epitope, but not the 3D10 epitope, to elicit functionally inhibitory anti-DBPII serum antibodies that blocked DBPII-erythrocyte binding.…”

Triosephosphate isomerase of Taenia solium (TTPI): phage display and antibodies as tools for finding target regions to inhibit catalytic activity

“…The most important feature lies in developing vaccines and efforts to obtain effective therapies for pathogens, such as Plasmodium vivax (Demangel et al 1998), Sc. mansoni (Arnon et al 2000), Cryptococcus neoformans (Fleuridor et al 2001), P. falciparum (Casey et al 2004;Sabo et al 2007), Fasciola hepatica (Villa-Mancera et al 2008, 2011, Streptococcus pneumonia (Smith et al 2009), Toxoplasma gondii (Cunha-Júnior et al 2010), and Helicobacter pylori (Li et al 2010).…”

Heterologous expression of Plasmodium vivax apical membrane antigen 1 (PvAMA1) for binding peptide selection

“…If natural peptide library is used in selection, antigenic peptide is called epitope, while mimotope is selected from the random peptide library. Epitope mapping using PfAMA1 peptide library ( Coley et al, 2001 ; Coley et al, 2006 ) as well as mimotopes mapping using random peptide library ( Casey et al, 2004 ; Sabo et al, 2007 ) against monoclonal anti-AMA1 antibodies (e.g., 5G8, 4G2dc1, and 1F9) that blocks merozoite invasion by P. falciparum have facilitated greater understanding of immune responses against AMA1. Several peptides that specifically bind to PfAMA1 and showed similar functionality to native anti-AMA1 have been isolated from random peptides libraries ( Li et al, 2002 ; Harris et al, 2005 ).…”

“…Nowadays, applications of both phage display technique together with the 3D structuring and/or protein docking techniques have sped up the studies on molecular binding interactions. For instance, the AMA1-binding molecules, i.e., monoclonal antibodies (both the epitopes and mimotopes) and peptides selected from phage display libraries, all of which block merozoite invasion of erythrocytes, were successfully characterized and have been mapped to the location on AMA1 ectoplasmic region by nuclear magnetic resonance spectroscopy ( Keizer et al, 2003 ; Sabo et al, 2007 ; Harris et al, 2009 ; Wang et al, 2014 ) and X-ray crystallographic analysis ( Bai et al, 2005 ; Pizarro et al, 2005 ). Overall, all the studies described above focused on PfAMA1 and this is the first study carried out on PvAMA1.…”