Identification of a Highly Antigenic Linear B Cell Epitope within Plasmodium vivax Apical Membrane Antigen 1 (AMA-1)
Lilian Lacerda BuenoFrancisco Pereira LoboCristiane Guimarães MoraisLuíza Carvalho MourãoRicardo Andrez Machado‐de‐ÁvilaIrene S. SoaresCor Jésus Fernandes FontesMarcus LacerdaCarlos Chávez-OlórteguiDaniella Castanheira BartholomeuRicardo Toshio FujiwaraÉrika Martins Braga
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Abstract:
Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290–307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies.Keywords:
Antigenicity
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Antigenic determinants, also called epitopes, constitute the region of the protein in intimate contact with the antigen-binding region of an antibody. Epitopes are typically classified as being either continuous or discontinuous. A variety of methods have been applied to the study of monoclonal antibody (mAb)-antigen interactions and the characterization of their respective epitopes. Here we introduce some new methods of peptide and protein epitope mapping by mass spectrometry.
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CD20, which is expressed on B lymphocytes, has been studied as a therapeutic target for B cell lymphomas and autoimmune disorders. Identifying the binding epitopes of monoclonal antibodies (mAbs) can contribute to our understanding of their functions. We have previously developed an anti-CD20 mAb (clone C20Mab-11) using a Cell-Based Immunization and Screening (CBIS) method. In this study, we aimed to determine the binding epitopes of anti-CD20 mAbs, such as C20Mab-11 and 2H7, using the His-tag insertion for epitope mapping (HisMAP). The results showed that 171-NPSE-174 and 168-EPANPSE-174 in the second loop of CD20 were essential for C20Mab-11 binding and 2H7 binding, respectively. Although we developed many mAbs that recognize conformational epitopes using the CBIS method, there are many difficulties in epitope mapping for these mAbs. HisMAP could be useful for determining the conformational epitopes of other mAbs against membrane proteins.
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An epitope can be simply defined as that part of an antigen involved in its recogmtion by an antibody In the case of protein antigens, an epitope would consist of a group of mdlvidual ammo-acid side-chains close together on the protein surface Epitope mapping, then, becomes the process of locating the epitope, or identifying the mdividual ammo-acids mvolved Apart from its mtrmsic value for understanding protein structure-function relationships, it also has a practical value in generating antibody probes of defined specificity as research tools and in helping to define the immune response to pathogenic proteins and organisms Some authors have even extended the epitope concept to the mteraction between peptide hormones and then receptors (1), not every immunologist would be happy about this, but it does make the point that in mapping epitopes, we are studying a biological process of fundamental importance, that of protein-protein mteraction Epitope mapping is usually done with monoclonal antibodies (MAbs), though it can be done with polyclonal antisera in a rather less rigorous way, bearing in mind that antisera behave as mixtures of MAbs. Mapping can be done directly by X-ray crystallography of antibody-antigen complexes, but it can also be done by changing individual ammo-acids, by using antigen fragments and synthetic peptides or by competition methods in which two or more antibodies compete for the same, or adjacent, epitopes. The term "epitope mapping" has also been used to describe the attempt to determine all the major sites on a protein surface that can elicit an antibody response, at the end of which one might claim to have produced an "epitope map" of the protein mununogen (2) This mformatron might be very useful, for example, to someone wishing to produce antiviral vaccines. However, there is a limit to how far one can go down this road, because the map obtained may be influenced by how MAbs are selected and by the mapping method used Furthermore, the more strictly correct definmon of epltope mapping is based on antigenicity (the ability to recognize a specific antibody).
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Antibodies play essential roles in both diagnostics and therapeutics. Epitope mapping is essential to understand how an antibody works and to protect intellectual property. Given the millions of antibodies for which epitope information is lacking, there is a need for high-throughput epitope mapping. To address this, we developed a strategy, Antibody binding epitope Mapping (AbMap), by combining a phage displayed peptide library with next-generation sequencing. Using AbMap, profiles of the peptides bound by 202 antibodies were determined in a single test, and linear epitopes were identified for >50% of the antibodies. Using spike protein (S1 and S2)-enriched antibodies from the convalescent serum of one COVID-19 patient as the input, both linear and potentially conformational epitopes of spike protein specific antibodies were identified. We defined peptide-binding profile of an antibody as the binding capacity (BiC). Conceptually, the BiC could serve as a systematic and functional descriptor of any antibody. Requiring at least one order of magnitude less time and money to map linear epitopes than traditional technologies, AbMap allows for high-throughput epitope mapping and creates many possibilities.
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Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-γ. Based on the mAbs' (n = 12) ability to simultaneously bind hIFN-γ in ELISA, 2 epitope clusters with 5 mAbs in each were defined; 2 mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-γ, epitopes were identified using 7 h/bIFN-γ chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-γ residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2 mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN-γ-mediated activation of human cells, in line with the involvement of region A in the IFN-γ receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes.
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