Overcoming Antigenic Diversity by Enhancing the Immunogenicity of Conserved Epitopes on the Malaria Vaccine Candidate Apical Membrane Antigen-1

Malaria vaccine candidate Apical Membrane Antigen-1 (AMA1) induces protection, but only against parasite strains that are closely related to the vaccine. Overcoming the AMA1 diversity problem will require an understanding of the structural basis of cross-strain invasion inhibition. A vaccine containing four diverse allelic proteins 3D7, FVO, HB3 and W2mef (AMA1 Quadvax or QV) elicited polyclonal rabbit antibodies that similarly inhibited the invasion of four vaccine and 22 non-vaccine strains of P. falciparum. Comparing polyclonal anti-QV with antibodies against a strain-specific, monovalent, 3D7 AMA1 vaccine revealed that QV induced higher levels of broadly inhibitory antibodies which were associated with increased conserved face and domain-3 responses and reduced domain-2 response. Inhibitory monoclonal antibodies (mAb) raised against the QV reacted with a novel cross-reactive epitope at the rim of the hydrophobic trough on domain-1; this epitope mapped to the conserved face of AMA1 and it encompassed the 1e-loop. MAbs binding to the 1e-loop region (1B10, 4E8 and 4E11) were ∼10-fold more potent than previously characterized AMA1-inhibitory mAbs and a mode of action of these 1e-loop mAbs was the inhibition of AMA1 binding to its ligand RON2. Unlike the epitope of a previously characterized 3D7-specific mAb, 1F9, the 1e-loop inhibitory epitope was partially conserved across strains. Another novel mAb, 1E10, which bound to domain-3, was broadly inhibitory and it blocked the proteolytic processing of AMA1. By itself mAb 1E10 was weakly inhibitory but it synergized with a previously characterized, strain-transcending mAb, 4G2, which binds close to the hydrophobic trough on the conserved face and inhibits RON2 binding to AMA1. Novel inhibition susceptible regions and epitopes, identified here, can form the basis for improving the antigenic breadth and inhibitory response of AMA1 vaccines. Vaccination with a few diverse antigenic proteins could provide universal coverage by redirecting the immune response towards conserved epitopes.     

Rapid and precise epitope mapping of monoclonal antibodies against Plasmodium falciparum AMA1 by combined phage display of fragments and random peptides.

We describe an approach for the rapid mapping of epitopes within a malaria antigen using a combination of phage display techniques. Phage display of antigen fragments identifies the location of the epitopes, then random peptide libraries displayed on phage are employed to identify accurately amino acids involved in the epitope. Finally, phage display of mutant fragments confirms the role of each residue in the epitope. This approach was applied to the apical membrane antigen-1 (AMA1), which is a leading candidate for inclusion in a vaccine directed against the asexual blood stages of Plasmodium falciparum. As part of the effort both to understand the function of AMA1 in the parasite life cycle and to define the specificity of protective immune responses, a panel of monoclonal antibodies (MAbs) was generated to obtain binding reagents to the various domains within the molecule. There is a pressing need to determine rapidly the regions recognized by these antibodies and the structural requirements required within AMA1 for high affinity binding of the MAbs. Using phage displaying random AMA1 fragments, it was shown that MAb5G8 recognizes a short linear epitope within the pro-domain of AMA1 whereas the epitope recognized by MAb 1F9 is reduction sensitive and resides within a disulphide-bonded 57 amino acid sub-domain of domain-1. Phage displaying random peptide libraries and mutant AMA1 fragments were employed for fine mapping of the MAb5G8 core epitope to a three-residue sequence in the AMA1 prodomain.     

Antibodies to Polymorphic Invasion-Inhibitory and Non-Inhibitory Epitopes of Plasmodium falciparum Apical Membrane Antigen 1 in Human Malaria

Background

Antibodies to P. falciparum apical membrane protein 1 (AMA1) may contribute to protective immunity against clinical malaria by inhibiting blood stage growth of P. falciparum, and AMA1 is a leading malaria vaccine candidate. Currently, there is limited knowledge of the acquisition of strain-specific and cross-reactive antibodies to AMA1 in humans, or the acquisition of invasion-inhibitory antibodies to AMA1.

Methodology/Findings

We examined the acquisition of human antibodies to specific polymorphic invasion-inhibitory and non-inhibitory AMA1 epitopes, defined by the monoclonal antibodies 1F9 and 2C5, respectively. Naturally acquired antibodies were measured in cohorts of Kenyan children and adults. Antibodies to the invasion-inhibitory 1F9 epitope and non-inhibitory 2C5 epitope were measured indirectly by competition ELISA. Antibodies to the 1F9 and 2C5 epitopes were acquired by children and correlated with exposure, and higher antibody levels and prevalence were observed with increasing age and with active P. falciparum infection. Of note, the prevalence of antibodies to the inhibitory 1F9 epitope was lower than antibodies to AMA1 or the 2C5 epitope. Antibodies to AMA1 ectodomain, the 1F9 or 2C5 epitopes, or a combination of responses, showed some association with protection from P. falciparum malaria in a prospective longitudinal study. Furthermore, antibodies to the invasion-inhibitory 1F9 epitope were positively correlated with parasite growth-inhibitory activity of serum antibodies.

Conclusions/Significance

Individuals acquire antibodies to functional, polymorphic epitopes of AMA1 that may contribute to protective immunity, and these findings have implications for AMA1 vaccine development. Measuring antibodies to the 1F9 epitope by competition ELISA may be a valuable approach to assessing human antibodies with invasion-inhibitory activity in studies of acquired immunity and vaccine trials of AMA1.     

Structure of an IgNAR-AMA1 complex: targeting a conserved hydrophobic cleft broadens malarial strain recognition

Apical membrane antigen 1 (AMA1) is essential for invasion of erythrocytes and hepatocytes by Plasmodium parasites and is a leading malarial vaccine candidate. Although conventional antibodies to AMA1 can prevent such invasion, extensive polymorphisms within surface-exposed loops may limit the ability of these AMA1-induced antibodies to protect against all parasite genotypes. Using an AMA1-specific IgNAR single-variable-domain antibody, we performed targeted mutagenesis and selection against AMA1 from three P. falciparum strains. We present cocrystal structures of two antibody-AMA1 complexes which reveal extended IgNAR CDR3 loops penetrating deep into a hydrophobic cleft on the antigen surface and contacting residues conserved across parasite species. Comparison of a series of affinity-enhancing mutations allowed dissection of their relative contributions to binding kinetics and correlation with inhibition of erythrocyte invasion. These findings provide insights into mechanisms of single-domain antibody binding, and may enable design of reagents targeting otherwise cryptic epitopes in pathogen antigens.     

Solution NMR characterization of apical membrane antigen 1 and small molecule interactions as a basis for designing new antimalarials

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti‐apical membrane antigen 1 antibodies and other inhibitory molecules that target this hydrophobic cleft are able to block the invasion process, PfAMA1 is an attractive target for the development of strain‐transcending antimalarial agents. As solution nuclear magnetic resonance spectroscopy is a valuable technique for the rapid characterization of protein–ligand interactions, we have determined the sequence‐specific backbone assignments for PfAMA1 from two P. falciparum strains, FVO and 3D7. Both selective labelling and unlabelling strategies were used to complement triple‐resonance experiments in order to facilitate the assignment process. We have then used these assignments for mapping the binding sites for small molecules, including benzimidazoles, pyrazoles and 2‐aminothiazoles, which were selected on the basis of their affinities measured from surface plasmon resonance binding experiments. Among the compounds tested, benzimidazoles showed binding to a similar region on both FVO and 3D7 PfAMA1, suggesting that these compounds are promising scaffolds for the development of novel PfAMA1 inhibitors. Copyright © 2016 John Wiley & Sons, Ltd.     

Cross-reactivity studies of an anti-Plasmodium vivax apical membrane antigen 1 monoclonal antibody: binding and structural characterisation

Apical membrane antigen 1 (AMA1) has an important, but as yet uncharacterised, role in host cell invasion by the malaria parasite, Plasmodium. The protein, which is quite conserved between Plasmodium species, comprises an ectoplasmic region, a single transmembrane segment and a small cytoplasmic domain. The ectoplasmic region, which can induce protective immunity in animal models of human malaria, is a leading vaccine candidate that has entered clinical trials. The monoclonal antibody F8.12.19, raised against the recombinant ectoplasmic region of AMA1 from Plasmodium vivax, cross-reacts with homologues from Plasmodium knowlesi, Plasmodium cynomolgi, Plasmodium berghei and Plasmodium falciparum, as shown by immunofluorescence assays on mature schizonts. The binding of F8.12.19 to recombinant AMA1 from both P. vivax and P. falciparum was measured by surface plasmon resonance, revealing an apparent affinity constant that is about 100-fold weaker for the cross-reacting antigen when compared to the cognate antigen. Crystal structure analysis of Fab F8.12.19 complexed to AMA1 from P. vivax and P. falciparum shows that the monoclonal antibody recognises a discontinuous epitope located on domain III of the ectoplasmic region, the major component being a loop containing a cystine knot. The structures provide a basis for understanding the cross-reactivity. Antibody contacts are made mainly to main-chain and invariant side-chain atoms of AMA1; contact antigen residues that differ in sequence are located at the periphery of the antigen-binding site and can be accommodated at the interface between the two components of the complex. The implications for AMA1 vaccine development are discussed.     

Molecular Insights into the Interaction between Plasmodium falciparum Apical Membrane Antigen 1 and an Invasion-Inhibitory Peptide

Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum has been implicated in invasion of the host erythrocyte. It interacts with malarial rhoptry neck (RON) proteins in the moving junction that forms between the host cell and the invading parasite. Agents that block this interaction inhibit invasion and may serve as promising leads for anti-malarial drug development. The invasion-inhibitory peptide R1 binds to a hydrophobic cleft on AMA1, which is an attractive target site for small molecules that block parasite invasion. In this work, truncation and mutational analyses show that Phe5-Phe9, Phe12 and Arg15 in R1 are the most important residues for high affinity binding to AMA1. These residues interact with two well-defined binding hot spots on AMA1. Computational solvent mapping reveals that one of these hot spots is suitable for small molecule targeting. We also confirm that R1 in solution binds to AMA1 with 1∶1 stoichiometry and adopts a secondary structure consistent with the major form of R1 observed in the crystal structure of the complex. Our results provide a basis for designing high affinity inhibitors of the AMA1-RON2 interaction.     

Structural consensus among antibodies defines the antigen binding site

The Complementarity Determining Regions (CDRs) of antibodies are assumed to account for the antigen recognition and binding and thus to contain also the antigen binding site. CDRs are typically discerned by searching for regions that are most different, in sequence or in structure, between different antibodies. Here, we show that ~20% of the antibody residues that actually bind the antigen fall outside the CDRs. However, virtually all antigen binding residues lie in regions of structural consensus across antibodies. Furthermore, we show that these regions of structural consensus which cover the antigen binding site are identifiable from the sequence of the antibody. Analyzing the predicted contribution of antigen binding residues to the stability of the antibody-antigen complex, we show that residues that fall outside of the traditionally defined CDRs are at least as important to antigen binding as residues within the CDRs, and in some cases, they are even more important energetically. Furthermore, antigen binding residues that fall outside of the structural consensus regions but within traditionally defined CDRs show a marginal energetic contribution to antigen binding. These findings allow for systematic and comprehensive identification of antigen binding sites, which can improve the understanding of antigenic interactions and may be useful in antibody engineering and B-cell epitope identification.     

Epitope specificity of hemagglutinating monoclonal anti-A-antibodies]

Fine epitope specificity of three anti-A monoclonal antibodies (MA) 1H410, 3F9, and 44F9 was studied by: 1) direct MA binding to synthetic oligosaccharides (OS) linked to polyacrylamide matrix, and 2) inhibition of MA binding to natural antigen by synthetic OS and their polyacrylamide conjugates. It has been established that the antigen binding site of MA 1H10 is specific for tetrasaccharide A (type 3), whereas MAs 3F9 and 44F9 recognize trisaccharide A, the contribution of alpha-L-fucosyl residue being insignificant in the case of 44F9 binding. The correlation of the MAs epitope specificity with their ability to agglutinate red blood cells of A1 and weak A subgroups is discussed.     

Structures of phage-display peptides that bind to the malarial surface protein,apical membrane antigen 1, and block erythrocyte invasion

Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum is synthesized by schizont stage parasites and has been implicated in merozoite invasion of host erythrocytes. Phage-display techniques have recently been used to identify two 15-residue peptides, F1 and F2, which bind specifically to P. falciparum AMA1 and inhibit parasite invasion of erythrocytes [Li, F., et al. (2002) J. Biol. Chem. 277, 50303-50310]. We have synthesized F1, F2, and three peptides with high levels of sequence identity, determined their relative binding affinities for P. falciparum AMA1 with a competition ELISA, and investigated their solution structures by NMR spectroscopy. The strongest binding peptide, F1, contains a beta-turn that includes residues identified via an alanine scan as being critical for binding to AMA1 and inhibition of merozoite invasion of erythrocytes. The three F1 analogues include a 10-residue analogue of F1 truncated at the C-terminus (tF1), a partially scrambled 15-mer (sF1), and a disulfide-constrained 14-mer (F1tbp) which is related to F1 but has a sequence identical to that of a disulfide-constrained loop in the first epidermal growth factor module of the latent transforming growth factor-beta binding protein. tF1 and F1tbp bound competitively with F1 to AMA1, and all three contain a type I beta-turn encompassing key residues involved in F1 binding. In contrast, sF1 lacked this structural motif, and did not compete for binding to AMA1 with F1; rather, sF1 contained a type III beta-turn involving a different part of the sequence. Although F2 was able to bind to AMA1, it was unstructured in solution, consistent with its weak invasion inhibitory effects. Thus, the secondary structure elements observed for these peptides in solution correlate well with their potency in binding to AMA1 and inhibiting merozoite invasion. The structures provide a valuable starting point for the development of peptidomimetics as antimalarial antagonists directed at AMA1.     

Fine specificity of the human immune response to the major neutralization epitopes expressed on cytomegalovirus gp58/116 (gB), as determined with human monoclonal antibodies.

The humoral immune response to human cytomegalovirus (CMV) membrane glycoprotein gp58/116 (gB) has been studied by establishing cell lines producing specific human monoclonal antibodies. These cell lines were generated from peripheral blood lymphocytes obtained from a healthy carrier. Hybridomas producing gp58/116-specific antibodies were detected by reactivity to procaryotically expressed proteins containing the major neutralizing epitopes of this glycoprotein complex. One antibody, ITC88, which recognized an epitope located between amino acid residues 67 and 86 of gp116, potently neutralized the virus at 1 to 2 micrograms of immunoglobulin G per ml. Only four of the six human antibodies detecting the major neutralizing domain of gp58 neutralized the virus, and none of them required complement for activity. All antibodies that bound mature, processed gp58 recognized a conformational epitope involving sequences between residues 549 and 635. However, small differences existed between the antibodies in the actual minimal requirement for C- and N-terminal parts of this epitope. By peptide mapping with several of the antibodies, the epitope was shown to consist mainly of residues between amino acids 570 to 579 and 606 to 619. Despite the conformational nature of the epitope, the antibodies recognized both reduced and denatured native antigen. Presence of carbohydrates was not required for antigen binding of these gp58-specific human antibodies, but in at least one case, it greatly enhanced antigen recognition, indicating an importance of carbohydrate structures in some epitopes within the major neutralizing specificity of gp58.     

Human immunodeficiency virus type 1-neutralizing monoclonal antibody 2F5 is multispecific for sequences flanking the DKW core epitope

Human monoclonal antibody 2F5 is one of a few human antibodies that neutralize a broad range of HIV-1 primary isolates. The 2F5 epitope on gp41 includes the sequence ELDKWA, with the core residues, DKW, being critical for antibody binding. HIV-neutralizing antibodies have never been elicited by immunization with peptides bearing ELDKWA, suggesting that important part(s) of the 2F5 paratope remain unidentified. The use of longer peptides extending beyond ELDKWA has resulted in increased epitope antigenicity, but neutralizing antibodies have not been generated. We sought to develop peptides that bind to 2F5, and that function as specific probes of the 2F5 paratope. Thus, we used 2F5 to screen a set of phage-displayed, random peptide libraries. Tight-binding clones from the random peptide libraries displayed sequence variability in the regions flanking the DKW motif. To further reveal flanking regions involved in 2F5 binding, two semi-defined libraries were constructed having 12 variegated residues either N-terminal or C-terminal to the DKW core (X(12)-AADKW and AADKW-X(12), respectively). Three clones isolated from the AADKW-X(12) library had similar high affinities, despite a lack of sequence homology among them, or with gp41. The contribution of each residue of these clones to 2F5 binding was evaluated by Ala substitution and amino acid deletion studies, and revealed that each clone bound 2F5 by a different mechanism. These results suggest that the 2F5 paratope is formed by at least two functionally distinct regions: one that displays specificity for the DKW core epitope, and another that is multispecific for sequences C-terminal to the core epitope. The implications of this second, multispecific region of the 2F5 paratope for its unique biological function are discussed.     

Peptide inhibitors of the malaria surface protein, apical membrane antigen 1: identification of key binding residues

Apical membrane antigen 1 (AMA1) is essential for malaria parasite invasion of erythrocytes and is therefore an attractive target for drug development. Peptides that bind AMA1 have been identified from random peptide libraries expressed on the surface of phage. Of these, R1, which binds to a hydrophobic ligand binding site on AMA1, was a particularly potent inhibitor of parasite invasion of erythrocytes in vitro. The solution structure of R1 contains a turn-like conformation between residues 5-10. Here the importance of residues in this turn-like structure for binding to AMA1 was examined by site-directed mutagenesis and NMR spectroscopy. The peptide was expressed as a fusion protein following replacement of Met16 by Leu in order to accommodate cyanogen bromide cleavage. This modified peptide (R2) displayed the same affinity for AMA1 as R1, showing that the identity of the side chain at position 16 was not critical for binding. Substitution of Phe5, Pro7, Leu8, and Phe9 with alanine led to significant (7.5- to >350-fold) decreases in affinity for AMA1. Comparison of backbone amide and C(α) H chemical shifts for these R2 analogues with corresponding values for R2 showed no significant changes, with the exception of R2(P7A), where slightly larger differences were observed, particularly for residues flanking position 7. The absence of significant changes in the secondary chemical shifts suggests that these mutations had little effect on the solution conformation of R2. The identification of a nonpolar region of these peptides containing residues essential for AMA1 binding establishes a basis for the design of anti-malarial drugs based on R1 mimetics.     

Identification of the discontinuous epitope in human complement protein C9 recognized by anti-melittin antibodies

Polyclonal rabbit antibodies against melittin recognize human C protein C9 and retard C9-mediated hemolysis. Human C9 contains a tetrameric and a pentameric sequence (amino acids 293-296 and 528-532, respectively) that together match a continuous segment in the melittin sequence, i.e., residues 8-16. It has been suggested that the tetrameric and the pentameric regions on C9 form a discontinuous epitope on folded C9 that mimics the structure of melittin. To further test this hypothesis, antibodies to C9-sequence-specific peptides were prepared. Peptides containing either the homologous tetrameric or the homologous pentameric sequence together with short stretches of the respective amino- and carboxyl-terminal flanking regions were synthesized, as well as a composite peptide predicted to resemble the discontinuous epitope as a linear, nine-amino acid sequence. Direct and competitive binding assays demonstrated that the tetrameric and the pentameric sequences are part of the epitope on human C9 that is recognized by anti-melittin IgG. However, only antibodies directed against the complete epitope are capable of inhibiting hemolysis. Because neither anti-tetramer nor anti-pentamer antibodies affect hemolysis whereas anti-melittin and anti-composite antibodies do, we propose that human C9 changes conformation around a hinge located between residues 296 and 528 and that the latter two antibodies inhibit unfolding required for membrane insertion and subsequent hemolysis.     

Structure and mechanistic analysis of the anti-human immunodeficiency virus type 1 antibody 2F5 in complex with its gp41 epitope

The membrane-proximal region of the ectodomain of the gp41 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is the target of three of the five broadly neutralizing anti-HIV-1 antibodies thus far isolated. We have determined crystal structures of the antigen-binding fragment for one of these antibodies, 2F5, in complex with 7-mer, 11-mer, and 17-mer peptides of the gp41 membrane-proximal region, at 2.0-, 2.1-, and 2.2-A resolutions, respectively. The structures reveal an extended gp41 conformation, which stretches over 30 A in length. Contacts are made with five complementarity-determining regions of the antibody as well as with nonpolymorphic regions. Only one exclusive charged face of the gp41 epitope is bound by 2F5, while the nonbound face, which is hydrophobic, may be hidden due to occlusion by other portions of the ectodomain. The structures reveal that the 2F5 antibody is uniquely built to bind to an epitope that is proximal to a membrane surface and in a manner mostly unaffected by large-scale steric hindrance. Biochemical studies with proteoliposomes confirm the importance of lipid membrane and hydrophobic context in the binding of 2F5 as well as in the binding of 4E10, another broadly neutralizing antibody that recognizes the membrane-proximal region of gp41. Based on these structural and biochemical results, immunization strategies for eliciting 2F5- and 4E10-like broadly neutralizing anti-HIV-1 antibodies are proposed.     

Influenza Human Monoclonal Antibody 1F1 Interacts with Three Major Antigenic Sites and Residues Mediating Human Receptor Specificity in H1N1 Viruses

Most monoclonal antibodies (mAbs) to the influenza A virus hemagglutinin (HA) head domain exhibit very limited breadth of inhibitory activity due to antigenic drift in field strains. However, mAb 1F1, isolated from a 1918 influenza pandemic survivor, inhibits select human H1 viruses (1918, 1943, 1947, and 1977 isolates). The crystal structure of 1F1 in complex with the 1918 HA shows that 1F1 contacts residues that are classically defined as belonging to three distinct antigenic sites, Sa, Sb and Ca₂. The 1F1 heavy chain also reaches into the receptor binding site (RBS) and interacts with residues that contact sialoglycan receptors and determine HA receptor specificity. The 1F1 epitope is remarkably similar to the previously described murine HC63 H3 epitope, despite significant sequence differences between H1 and H3 HAs. Both antibodies potently inhibit receptor binding, but only HC63 can block the pH-induced conformational changes in HA that drive membrane fusion. Contacts within the RBS suggested that 1F1 may be sensitive to changes that alter HA receptor binding activity. Affinity assays confirmed that sequence changes that switch the HA to avian receptor specificity affect binding of 1F1 and a mAb possessing a closely related heavy chain, 1I20. To characterize 1F1 cross-reactivity, additional escape mutant selection and site-directed mutagenesis were performed. Residues 190 and 227 in the 1F1 epitope were found to be critical for 1F1 reactivity towards 1918, 1943 and 1977 HAs, as well as for 1I20 reactivity towards the 1918 HA. Therefore, 1F1 heavy-chain interactions with conserved RBS residues likely contribute to its ability to inhibit divergent HAs.     

Phage display and peptide mapping of an immunoglobulin light chain fibril-related conformational epitope

Amyloid fibrils and partially unfolded intermediates can be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we previously had reported that mAb 11-1F4, generated by immunizing mice with a thermally denatured variable domain (VL) fragment of the human kappa4 Bence Jones protein Len, bound to a non-native conformational epitope located within the N-terminal 18 residues of fibrillar, as well as partially denatured, Ig light chains (O'Nuallain, B., et al. (2006) Biochemistry 46, 1240-1247). To define further the antibody binding site, we used random peptide phage display and epitope mapping of VL Len using wild-type and alanine-mutated Len peptides where it was shown that the antibody epitope was reliant on up to 10 of the first 15 residues of protein Len. Comparison of Vkappa and Vlambda N-terminal germline consensus sequences with protein Len and 11-1F4-binding phages indicated that this antibody's cross-reactivity with light chains was related to an invariant proline at position(s) 7 and/or 8, bulky hydrophobic residues at positions 11 and 13, and additionally, to the ability to accommodate amino acid diversity at positions 1-4. Sequence alignments of the phage peptides revealed a central proline, often flanked by aromatic residues. Taken together, these results have provided evidence for the structural basis of the specificity of 11-1F4 for both kappa and lambda light chain fibrils. We posit that the associated binding site involves a rare type VI beta-turn or touch-turn that is anchored by a cis-proline residue. The identification of an 11-1F4-related mimotope should facilitate development of pan-light chain fibril-reactive antibodies that could be used in the diagnosis and treatment of patients with AL amyloidosis.     

Structural Basis of Epitope Recognition by Heavy-Chain Camelid Antibodies

Truncated versions of heavy-chain antibodies (HCAbs) from camelids, also termed nanobodies, comprise only one-tenth the mass of conventional antibodies, yet retain similar, high binding affinities for the antigens. Here we analyze a large data set of nanobody–antigen crystal structures and investigate how nanobody–antigen recognition compares to the one by conventional antibodies. We find that nanobody paratopes are enriched in aromatic residues just like conventional antibodies, but additionally, they also bear a more hydrophobic character. Most striking differences were observed in the characteristics of the antigen's epitope. Unlike conventional antibodies, nanobodies bind to more rigid, concave, conserved and structured epitopes enriched with aromatic residues. Nanobodies establish fewer interactions with the antigens compared to conventional antibodies, and we speculate that high binding affinities are achieved due to less unfavorable conformational and more favorable solvation entropy contributions. We observed that interactions with antigen are mediated not only by three CDR loops but also by numerous residues from the nanobody framework. These residues are not distributed uniformly; rather, they are concentrated into four structurally distinct regions and mediate mostly charged interactions. Our findings suggest that in some respects nanobody–antigen interactions are more similar to the general protein–protein interactions rather than antibody–antigen interactions.     

Dissecting contact potentials for proteins: relative contributions of individual amino acids

Mimotopes mimic the three-dimensional topology of an antigen epitope, and are frequently recognized by antibodies with affinities comparable to those obtained for the original antibody-antigen interaction. Peptides and anti-idiotypic antibodies are two classes of protein mimotopes that mimic the topology (but not necessarily the sequence) of the parental antigen. In this study, we combine these two classes by selecting mimotopes based on single domain IgNAR antibodies, which display exceptionally long CDR3 loop regions (analogous to a constrained peptide library) presented in the context of an immunoglobulin framework with adjacent and supporting CDR1 loops. By screening an in vitro phage-display library of IgNAR variable domains (V(NAR)s) against the target antigen monoclonal antibody MAb5G8, we obtained four potential mimotopes. MAb5G8 targets a linear tripeptide epitope (AYP) in the flexible signal sequence of the Plasmodium falciparum Apical Membrane Antigen-1 (AMA1), and this or similar motifs were detected in the CDR loops of all four V(NAR)s. The V(NAR)s, 1-A-2, -7, -11, and -14, were demonstrated to bind specifically to this paratope by competition studies with an artificial peptide and all showed enhanced affinities (3-46 nM) compared to the parental antigen (175 nM). Crystallographic studies of recombinant proteins 1-A-7 and 1-A-11 showed that the SYP motifs on these V(NAR)s presented at the tip of the exposed CDR3 loops, ideally positioned within bulge-like structures to make contact with the MAb5G8 antibody. These loops, in particular in 1-A-11, were further stabilized by inter- and intra- loop disulphide bridges, hydrogen bonds, electrostatic interactions, and aromatic residue packing. We rationalize the higher affinity of the V(NAR)s compared to the parental antigen by suggesting that adjacent CDR1 and framework residues contribute to binding affinity, through interactions with other CDR regions on the antibody, though of course definitive support of this hypothesis will rely on co-crystallographic studies. Alternatively, the selection of mimotopes from a large (<4 x 10(8)) constrained library may have allowed selection of variants with even more favorable epitope topologies than present in the original antigenic structure, illustrating the power of in vivo selection of mimotopes from phage-displayed molecular libraries.     

<Emphasis Type="Italic">O</Emphasis>-GlcNAc modification of the anti-malarial vaccine candidate <Emphasis Type="Italic">Pf</Emphasis>AMA1: <Emphasis Type="Italic">in silico</Emphasis>-defined structural changes and potential to generate a better vaccine

The complex life cycle of plasmodial parasites makes the selection of a single subunit protein a less than optimal strategy to generate an efficient vaccinal protection against malaria. Moreover, the full protection afforded by malarial proteins carried by intact parasites implies that immune responses against different antigens expressed in different phases of the cycle are required, but also suggests that native malarial antigens are presented to the host immune system in a manner that recombinant proteins do not achieve. The malarial apical membrane antigen 1 (AMA1) represents a suitable vaccine candidate because AMA1 is expressed on sporozoites and merozoites and allows them to invade hepatocytes and erythrocytes, respectively. Anti-AMA1 antibodies and cytotoxic T-cells are therefore expected to interfere both with the primary invasion of hepatocytes by sporozoites and with the later propagation of merozoites in erythrocytes, and thus efficiently counteract parasite development in its human host. AMA1 bears potential glycosylation sites and the human erythrocytic O-linked N-acetylglucosamine transferase (OGT) could glycosylate AMA1 through combinatorial metabolism. This hypothesis was tested in silico by developing binding models of AMA1 with human OGT complexed with UDP-GlcNc, and followed by the binding of O-GlcNAc with the hydroxyl group of AMA1 serine and threonine residues. Our results suggests that AMA1 shows potential for glycosylation at Thr517 and Ser498 and that O-GlcNAc AMA1 may constitute a conformationally more appropriate antigen for developing a protective anti-malarial immune response.