The Specificity of 130-kDa Leech Sensory Afferent Proteins Is Encoded by Their Carbohydrate Epitopes

From early development through adulthood in the leech, sensory afferents, glial cells, and connective tissue express different epitopes located on a group of 130-kDa glycoproteins. The sensory epitope [reactive with monoclonal antibody (mAb) Lan3-2] is shared by the peripheral sensory afferents of different sensory modalities. In contrast, three other immunocytochemically distinct epitopes (reactive with mAbs Laz2-369, Laz7-79, and Laz6-212) differentiate these sensory afferents according to their sensory modalities. The glial epitope (mAb Laz6-297) is expressed on all macroglial processes, and the connective tissue epitope (mAb Laz9-84) is located on connective tissue surrounding the CNS, as well as in the peripheral tissues. The hydrophilic-hydrophobic nature of the 130-kDa sensory afferent and glial proteins was determined by phase separation with Triton X-114 and hypoosmotic extraction. They behave as peripheral membrane proteins. Deglycosylation of 130-kDa glycoproteins with N-Glycanase or preincubation of their respective mAbs with alpha-methylmannoside showed that the sensory epitope contains mannose, whereas the modality epitopes are of an undefined carbohydrate character. Immunoprecipitation and a peptide mapping experiment confirmed the existence of four distinct sensory afferent epitopes. Previous studies provided evidence that the mannose-containing Lan3-2 epitope mediates normal sensory afferent growth in the synaptic neuropile. We, therefore, postulate that the carbohydrate epitopes on sensory afferent glycoproteins participate in synapse formation.     

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Summary Phage peptide libraries constitute powerful tools for the mapping of epitopes recognized by monoclonal antibodies (mAbs). Using screening of phage displayed random peptide libraries we have characterized the binding epitopes of three mAbs directed against the surface envelope glycoprotein (gp46) of the human T-cell leukemia virus type I (HTLV-I). Two phage libraries, displaying random heptapeptides with or without flanking cysteine residues, were screened for binding to mAbs 7G5D8, DB4 and 4F5F6. The SSSSTPL consensus sequence isolated from constrained heptapeptide library defines the epitope recognized by DB4 mAb and corresponds to the exact region 249–252 of the virus sequence. The APPMLPH consensus sequence isolated from non constrained heptapeptide library defines the epitope recognized by 7G5D8 mAb and corresponds to the region 187–193 with a single amino acid substitution, methionine to leucine at position 190. The third consensus sequence LYWPHD isolated from constrained heptapeptide library defines the epitope recognized by 4F5F6 mAb. It corresponds to an epitope without direct equivalence with the virus sequence. The data presented here showed that 7G5D8 and DB4 mAbs are raised against linear epitopes while 4F5F6 mAb recognized a continoous topographic epitope.     

Subunit selectivity and epitope characterization of mAbs directed against the GABAA/benzodiazepine receptor

mAbs bd 17, bd 24, and bd 28 raised against bovine cerebral gamma-aminobutyric acid (GABAA)/benzodiazepine receptors were analyzed for their ability to detect each of 12 GABAA receptor subunits expressed in cultured mammalian cells. Results showed that mAb bd 17 recognizes epitopes on both beta 2 and beta 3 subunits while mAb bd 24 is selective for the alpha 1 subunit of human and bovine, but not of rat origin. The latter antibody reacts with the rat alpha 1 subunit carrying an engineered Leu at position four, documenting the first epitope mapping of a GABAA receptor subunit-specific mAb. In contrast to mAbs bd 17 and bd 24, mAb bd 28 reacts with all GABAA receptor subunits tested but not with a glycine receptor subunit, suggesting the presence of shared epitopes on subunits of GABA-gated chloride channels.     

Mapping of conformational mAb epitopes to the C domain of human angiotensin I-converting enzyme

Angiotensin I-converting enzyme (ACE, CD143) has two homologous domains, each having a functional active site. Fine epitope mapping of 8 mAbs to the C-terminal domain of human ACE was carried out using plate precipitation assays, mAbs' cross-reactivity with ACE from different species, site-directed mutagenesis, and antigen- and cell-based ELISAs. Almost all epitopes contained potential glycosylation sites. Therefore, these mAbs could be used to distinguish different glycoforms of ACE expressed in different tissues or cell lines. mAbs 1B8 and 3F10 were especially sensitive to the composition of the N-glycan attached to Asn 731; mAbs 2H9 and 3F11 detected the glycosylation status of the glycan attached to Asn 685 and perhaps Asn1162; and mAb 1E10 and 4E3 recognized the glycan on Asn 666. The epitope of mAb 1E10 is located at the N-terminal end of the C domain, close to the unique 36 amino acid residues of testicular ACE (tACE). Moreover, it binds preferentially to tACE on the surface of human spermatozoa and thus may find application as an immunocontraceptive drug. mAb 4E3 was the best mAb for quantification of ACE-expressing somatic cells by flow cytometry. In contrast to the other mAbs, binding of mAb 2B11 was not markedly influenced by ACE glycosylation or by the cell culture conditions or cell types, making this mAb a suitable reference antibody. Epitope mapping of these C-domain mAbs, particularly those that compete with N-domain mAbs, enabled us to propose a model of the two-domain somatic ACE that might explain the interdomain cooperativity. Our findings demonstrated that mAbs directed to conformational epitopes on the C-terminal domain of human ACE are very useful for the detection of testicular and somatic ACE, quantification using flow cytometry and ELISA assays, and for the study of different aspects of ACE biology.     

Mechanisms Mediating Enhanced Neutralization Efficacy of Staphylococcal Enterotoxin B by Combinations of Monoclonal Antibodies

Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-β chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used to validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Finally structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations.     

Phage display revisited: Epitope mapping of a monoclonal antibody directed against Neisseria meningitidis adhesin A using the PROFILER technology

There is a strong need for rapid and reliable epitope mapping methods that can keep pace with the isolation of increasingly larger numbers of mAbs. We describe here the identification of a conformational epitope using Phage-based Representation OF ImmunoLigand Epitope Repertoire (PROFILER), a recently developed high-throughput method based on deep sequencing of antigen-specific lambda phage-displayed libraries. A novel bactericidal monoclonal antibody (mAb 9F11) raised against Neisseria meningitidis adhesin A (NadA), an important component of the Bexsero^® anti-meningococcal vaccine, was used to evaluate the technique in comparison with other epitope mapping methods. The PROFILER technology readily identified NadA fragments that were capable of fully recapitulating the reactivity of the entire antigen against mAb 9F11. Further analysis of these fragments using mutagenesis and hydrogen-deuterium exchange mass-spectrometry allowed us to identify the binding site of mAb 9F11 (A250-D274) and an adjoining sequence (V275-H312) that was also required for the full functional reconstitution of the epitope. These data suggest that, by virtue of its ability to detect a great variety of immunoreactive antigen fragments in phage-displayed libraries, the PROFILER technology can rapidly and reliably identify epitope-containing regions and provide, in addition, useful clues for the functional characterization of conformational mAb epitopes.     

Epitope mapping of Borrelia burgdorferi OspC protein in homodimeric fold

In current work, we used recombinant OspC protein derived from B. afzelii strain BRZ31 in the native homodimeric fold for mice immunization and following selection process to produce three mouse monoclonal antibodies able to bind to variable parts of up to five different OspC proteins. Applying the combination of mass spectrometry assisted epitope mapping and affinity based theoretical prediction we have localized regions responsible for antigen‐antibody interactions and approximate epitopes' amino acid composition. Two mAbs (3F4 and 2A9) binds to linear epitopes located in previously described immunogenic regions in the exposed part of OspC protein. The third mAb (2D1) recognises highly conserved discontinuous epitope close to the ligand binding domain 1.     

Fine and domain-level epitope mapping of botulinum neurotoxin type A neutralizing antibodies by yeast surface display

Botulinum neurotoxin (BoNT), the most poisonous substance known, causes naturally occurring human disease (botulism) and is one of the top six biothreat agents. Botulism is treated with polyclonal antibodies produced in horses that are associated with a high incidence of systemic reactions. Human monoclonal antibodies (mAbs) are under development as a safer therapy. Identifying neutralizing epitopes on BoNTs is an important step in generating neutralizing mAbs, and has implications for vaccine development. Here, we show that the three domains of BoNT serotype A (BoNT/A) can be displayed on the surface of yeast and used to epitope map six mAbs to the toxin domains they bind. The use of yeast obviates the need to express and purify each domain, and it should prove possible to display domains of other BoNT subtypes and serotypes for epitope mapping. Using a library of yeast-displayed BoNT/A binding domain (H(C)) mutants and selecting for loss of binding, the fine epitopes of three neutralizing BoNT/A mAbs were identified. Two mAbs bind the C-terminal subdomain of H(C), with one binding near the toxin sialoganglioside binding site. The most potently neutralizing mAb binds the N-terminal subdomain of H(C), in an area not previously thought to be functionally important. Modeling the epitopes shows how all three mAbs could bind BoNT/A simultaneously and may explain, in part, the dramatic synergy observed on in vivo toxin neutralization when these antibodies are combined. The results demonstrate how yeast display can be used for domain-level and fine mapping of conformational BoNT antibody epitopes and the mapping results identify three neutralizing BoNT/A epitopes.     

Epitope characterization of anti-JAM-A antibodies using orthogonal mass spectrometry and surface plasmon resonance approaches

Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells and associated with cancer progression. We present here the extensive characterization of immune complexes involving JAM-A antigen and three monoclonal antibodies (mAbs), including hz6F4-2, a humanized version of anti-tumoral 6F4 mAb identified by a functional and proteomic approach in our laboratory. A specific workflow that combines orthogonal approaches has been designed to determine binding stoichiometries along with JAM-A epitope mapping determination at high resolution for these three mAbs. Native mass spectrometry experiments revealed different binding stoichiometries and affinities, with two molecules of JAM-A being able to bind to hz6F4-2 and F11 Fab, while only one JAM-A was bound to J10.4. Surface plasmon resonance indirect competitive binding assays suggested epitopes located in close proximity for hz6F4-2 and F11. Finally, hydrogen-deuterium exchange mass spectrometry was used to precisely identify epitopes for all mAbs. The results obtained by orthogonal biophysical approaches showed a clear correlation between the determined epitopes and JAM-A binding characteristics, allowing the basis for molecular recognition of JAM-A by hz6F4-2 to be definitively established for the first time. Taken together, our results highlight the power of MS-based structural approaches for epitope mapping and mAb conformational characterization.     

Development of a humanized antibody with high therapeutic potential against dengue virus type 2

Background

Dengue virus (DENV) is a significant public health threat in tropical and subtropical regions of the world. A therapeutic antibody against the viral envelope (E) protein represents a promising immunotherapy for disease control.

Methodology/Principal Findings

We generated seventeen novel mouse monoclonal antibodies (mAbs) with high reactivity against E protein of dengue virus type 2 (DENV-2). The mAbs were further dissected using recombinant E protein domain I-II (E-DI-II) and III (E-DIII) of DENV-2. Using plaque reduction neutralization test (PRNT) and mouse protection assay with lethal doses of DENV-2, we identified four serotype-specific mAbs that had high neutralizing activity against DENV-2 infection. Of the four, E-DIII targeting mAb DB32-6 was the strongest neutralizing mAb against diverse DENV-2 strains. Using phage display and virus-like particles (VLPs) we found that residue K310 in the E-DIII A-strand was key to mAb DB32-6 binding E-DIII. We successfully converted DB32-6 to a humanized version that retained potency for the neutralization of DENV-2 and did not enhance the viral infection. The DB32-6 showed therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models even in post-exposure trials.

Conclusions/Significance

We used novel epitope mapping strategies, by combining phage display with VLPs, to identify the important A-strand epitopes with strong neutralizing activity. This study introduced potential therapeutic antibodies that might be capable of providing broad protection against diverse DENV-2 infections without enhancing activity in humans.     

Epitope mapping analysis of apolipoprotein B-100 using a surface plasmon resonance-based biosensor

Using a surface plasmon resonance (SPR)-based biosensor (BIA-technology), we have studied the interaction of ten different murine monoclonal antibodies (mAbs, all IgG₁), raised against the main protein constituent of human low density lipoprotein (LDL), i.e. the apolipoprotein B-100 (apoB-100). These mAbs identify distinct domains on apoB-100, relevant to LDL-receptor interaction: epitopes in the amino-terminal region (mAbs L7, L9, L10 and L11: aa 1–1297) and in the middle region (mAb 6B: aa 1480–1693; mAbs 2A, 3B: aa 2152–2377; mAbs 9A, L2 and L4: aa 2657–3248) of native apoB-100. A multisite binding analysis was performed to further characterize the epitopes recognized by all these mAbs. A rabbit anti-mouse IgG₁-Fc antibody (RAM.Fc) was first coupled to the gold surface in order to capture one anti-human apoB-100 mAb. ApoB-100 protein was subsequently injected and allowed to react with this immobilized, oriented antibody. Multisite binding assays were then performed, by sequentially flowing other mAbs, in different orders, over the sensing surface. The capacity of each mAb to interact with the entrapped apoB-100 in a multimolecular complex was monitored in real time by SPR. The results achieved were comparable to those obtained by western immunoblotting using the same reagents. However, SPR ensures a more detailed epitope identification, demonstrating that BIA-technology can be successfully used for mapping distinct epitopes on apoB-100 protein in solution dispensing with labels and secondary tracers; moreover, compared with conventional immunoassays, it is significantly time saving (CNR-P.F. MADESS 2).     

Determining the Binding Affinity of Therapeutic Monoclonal Antibodies towards Their Native Unpurified Antigens in Human Serum

Monoclonal antibodies (mAbs) are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen’s endogenous concentration, by combining the kinetic exclusion assay and Biacore’s calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9), progranulin (PGRN), and fatty acid binding protein (FABP4). We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.     

B细胞表位作图研究进展

抗原-抗体的特异性结合是由抗体表面的抗原决定簇与抗原表面的表位基序间的特异性互补识别决定的。B细胞表位作图既包括B细胞抗原表位基序的鉴定(即确定抗原分子上被B细胞表面受体或抗体特异性识别并结合的氨基酸基序),也包括绘制抗原蛋白的全部或接近全部的B细胞表位基序在其一级或高级结构上的分布图谱的过程。B细胞表位作图是研发表位疫苗、治疗性表位抗体药物和建立疾病免疫诊断方法的重要前提。目前,已经建立了多种B细胞表位鉴定或绘制抗原蛋白B细胞表位图谱的实验方法。基于抗原-单抗复合物晶体结构的X-射线晶体学分析的B细胞表位作图和基于抗原蛋白或抗原片段的突变体库筛选技术的B细胞表位作图可以在氨基酸水平,甚至原子水平上揭示抗原分子上与单抗特异性结合的关键基序;其它B细胞表位作图方法(如基于ELISA的肽库筛选技术)常常只能获得包含B细胞表位的抗原性肽段,因而,很少用于最小表位基序的鉴定;而改良的生物合成肽法多用于B细胞表位的最小基序鉴定和精细作图。鉴于每种B细胞作图方法都存在各自的优势与不足,B细胞表位作图往往需要多种作图方法的有机结合。本文对目前常用的B细胞表位作图的实验方法及其在动物疫病防控中的应用进行综述,以期为研究者设计最佳的表位作图方案提供参考。     

Mapping epitopes of monoclonal antibodies against HIV-1 integrase with limited proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Monoclonal antibodies (mAbs) have been used extensively in the biochemical analysis of proteins. Molecular identification of a specific epitope can enhance our understanding of the relationship between the structure and function of a protein. We recently developed a protein footprint technique for mapping mAb epitopes that employs limited proteolysis followed by peptide analysis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Here we describe the rational for the technique and illustrate its use in mapping the epitopes of two mAbs that bind to the C-terminal domain of human immunodeficiency virus type-1 integrase. The results provide a plausible explanation for the fact that one mAb inhibits enzyme activity while the second does not.     

Broad epitope coverage of a human in vitro antibody library

Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad “epitope coverage” increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or “bins” and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes.     

Engineered domain-based assays to identify individual antibodies in oligoclonal combinations targeting the same protein

Quantitation of individual monoclonal antibodies (mAbs) within a combined antibody drug product is required for preclinical and clinical drug development. We have developed two antitoxins, XOMA 3B and XOMA 3E, each consisting of three mAbs that neutralize type B and type E botulinum neurotoxin (BoNT/B and BoNT/E) to treat serotype B and E botulism. To develop mAb-specific binding assays for each antitoxin, we mapped the epitopes of the six mAbs. Each mAb bound an epitope on either the BoNT light chain (LC) or translocation domain (H_N). Epitope mapping data were used to design LC-H_N domains with orthogonal mutations to make them specific for only one mAb in either XOMA 3B or XOMA 3E. Mutant LC-H_N domains were cloned, expressed, and purified from Escherichia coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. Further engineering of domains allowed construction of enzyme-linked immunosorbent assays (ELISAs) that could characterize the integrity, binding affinity, and identity of each of the six mAbs in XOMA 3B and 3E without interference from the three BoNT/A mAbs in XOMA 3AB. Such antigen engineering is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that bind the same protein.     

Characterization of continuous B‐cell epitopes in the N‐terminus of glutamate decarboxylase67 using monoclonal antibodies

Glutamate decarboxylase (GAD) is an autoantigen associated with the autoimmune disorders Type‐1 diabetes (T1D) and stiff‐person syndrome (SPS). The protein, being an essential enzyme involved in the production of the inhibitory neurotransmitter γ‐aminobutyric acid, exists in two isoforms, GAD67 and GAD65. Both isoforms may be targeted by autoantibodies in SPS and T1D patients, although SPS primarily is associated with the presence of GAD67 autoantibodies, whereas T1D mainly is associated with the presence of GAD65 autoantibodies. In this study, we describe antibody reactivity to overlapping GAD67 peptides covering the complete protein sequence by modified peptide enzyme‐linked immunosorbent assay in order to identify potential GAD67 epitopes using two monoclonal antibodies (mAbs). Both GAD67 mAbs showed reactivity to linear epitopes located at the N‐terminal end of GAD67. The epitopes of GAD mAb 1 and 2 were identified as the amino acid sequences NAGADPNTTN and TETDFSNLF, respectively, corresponding to amino acids 14–23 and 91–99. Fine mapping of the epitopes revealed that antibody reactivity was related to amino acid side‐chain functionality, rather than amino acid side‐chain specificity. Additionally, results suggested that non‐contact amino acids in the epitope structure were essential for antibody reactivity. The exact role of these amino acids remains to be determined, but they are thought to be involved in backbone hydrogen bonds or stabilization of the epitope structure. As only limited knowledge is available in relation to antigenic regions of GAD67, this study contributes to characterization of GAD67 epitopes and may be a first step in the development of peptide‐based therapeutics against SPS. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

ABSTRACT: BACKGROUND: Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies. METHODS: Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20--57 years (median = 33 years) were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. RESULTS: We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL), suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. CONCLUSIONS: Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, in context of V3 region.     

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Phage peptide libraries constitute powerful tools for themapping of epitopes recognized by monoclonal antibodies (mAbs).Using screening of phage displayed random peptide libraries wehave characterized the binding epitopes of three mAbs directedagainst the surface envelope glycoprotein (gp46) of the humanT-cell leukemia virus type I (HTLV-I). Two phage libraries,displaying random heptapeptides with or without flankingcysteine residues, were screened for binding to mAbs 7G5D8, DB4and 4F5F6. The SSSSTPL consensus sequence isolated fromconstrained heptapeptide library defines the epitope recognizedby DB4 mAb and corresponds to the exact region 249–252 of thevirus sequence. The APPMLPH consensus sequence isolated fromnon constrained heptapeptide library defines the epitoperecognized by 7G5D8 mAb and corresponds to the region 187–193with a single amino acid substitution, methionine to leucine atposition 190. The third consensus sequence LYWPHD isolated fromconstrained heptapeptide library defines the epitope recognizedby 4F5F6 mAb. It corresponds to an epitope without directequivalence with the virus sequence. The data presented hereshowed that 7G5D8 and DB4 mAbs are raised against linearepitopes while 4F5F6 mAb recognized a continuous topographic epitope.     

Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display

Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat. Thus, understanding mechanisms of antibody-mediated viral inhibition and neutralization escape is critical. Here, a robust yeast display system for fine epitope mapping of viral surface hemagglutinin (HA)-specific antibodies is demonstrated. The full-length H5 subtype HA (HA0) was expressed on the yeast surface in a correctly folded conformation, determined by binding of a panel of extensively characterized neutralizing human monoclonal antibodies (mAbs). These mAbs target conformationally-dependent epitopes of influenza A HA, which are highly conserved across H5 clades and group 1 serotypes. By separately displaying HA1 and HA2 subunits on yeast, domain mapping of two anti-H5 mAbs, NR2728 and H5-2A, localized their epitopes to HA1. These anti-H5 mAb epitopes were further fine mapped by using a library of yeast-displayed HA1 mutants and selecting for loss of binding without prior knowledge of potential contact residues. By overlaying key mutant residues that impacted binding onto a crystal structure of HA, the NR2728 mAb was found to interact with a fully surface-exposed contiguous patch of residues at the receptor binding site (RBS), giving insight into the mechanism underlying its potent inhibition of virus binding. The non-neutralizing H5-2A mAb was similarly mapped to a highly conserved H5 strain-specific but poorly accessible location on a loop at the trimer HA interface. These data further augment our toolchest for studying HA antigenicity, epitope diversity and accessibility in response to natural and experimental influenza infection and vaccines.