Fine Epitope Mapping Research Articles

High-resolution epitope mapping of commercial antibodies to ANCA antigens by yeast surface display

Epitope mapping provides critical insight into antibody-antigen interactions. Epitope mapping of autoantibodies from patients with autoimmune diseases can help elucidate disease immunogenesis and guide the development of antigen-specific therapies. Similarly, epitope mapping of commercial antibodies targeting known autoantigens enables the use of those antibodies to test specific hypotheses. Anti-Neutrophil Cytoplasmic Autoantibody (ANCA) vasculitis results from the formation of autoantibodies to multiple autoantigens, including myeloperoxidase (MPO), proteinase-3 (PR3), plasminogen (PLG), and peroxidasin (PXDN). To perform high-resolution epitope mapping of commercial antibodies to these autoantigens, we developed a novel yeast surface display library based on a series of >5000 overlapping peptides derived from their protein sequences. Using both FACS and magnetic bead isolation of reactive yeast, we screened 19 commercially available antibodies to the ANCA autoantigens. This approach to epitope mapping resulted in highly specific, fine epitope mapping, down to single amino acid resolution in many cases. Our study also identified cross-reactivity between some commercial antibodies to MPO and PXDN, which suggests that patients with apparent autoantibodies to both proteins may be the result of cross-reactivity. Together, our data validate yeast surface display using maximally overlapping peptides as an excellent approach to linear epitope mapping.

Immunoanalytical Approach for Detecting and Identifying Ancestral Peptide Biomarkers in Early Earth Analogue Environments.

Several mass spectrometry and spectroscopic techniques have been used in the search for molecular biomarkers on Mars. A major constraint is their capability to detect and identify large and complex compounds such as peptides or other biopolymers. Multiplex immunoassays can detect these compounds, but antibodies must be produced for a large number of sequence-dependent molecular targets. Ancestral Sequence Reconstruction (ASR) followed by protein "resurrection" in the lab can help to narrow the selection of targets. Herein, we propose an immunoanalytical method to identify ancient and universally conserved protein/peptide sequences as targets for identifying ancestral biomarkers in nature. We have developed, tested, and validated this approach by producing antibodies to eight previously described ancestral resurrected proteins (three β-lactamases, three thioredoxins, one Elongation Factor Tu, and one RuBisCO, all of them theoretically dated as Precambrian), and used them as a proxy to search for any potential feature of them that could be present in current natural environments. By fluorescent sandwich microarray immunoassays (FSMI), we have detected positive immunoreactions with antibodies to the oldest β-lactamase and thioredoxin proteins (ca. 4 Ga) in samples from a hydrothermal environment. Fine epitope mapping and inhibitory immunoassays allowed the identification of well-conserved epitope peptide sequences that resulted from ASR and were present in the sample. We corroborated these results by metagenomic sequencing and found several genes encoding analogue proteins with significant matches to the peptide epitopes identified with the antibodies. The results demonstrated that peptides inferred from ASR studies have true counterpart analogues in Nature, which validates and strengthens the well-known ASR/protein resurrection technique and our immunoanalytical approach for investigating ancient environments and metabolisms on Earth and elsewhere.

Component-Resolved Diagnosis Based on a Recombinant Variant of Mus m 1 Lipocalin Allergen.

Exposure to the Mus m 1 aeroallergen is a significant risk factor for laboratory animal allergy. This allergen, primarily expressed in mouse urine where it is characterized by a marked and dynamic polymorphism, is also present in epithelium and dander. Considering the relevance of sequence/structure assessment in protein antigenic reactivity, we compared the sequence of the variant Mus m 1.0102 to other members of the Mus m 1 allergen, and used Discotope 2.0 to predict conformational epitopes based on its 3D-structure. Conventional diagnosis of mouse allergy is based on serum IgE testing, using an epithelial extract as the antigen source. Given the heterogeneous and variable composition of extracts, we developed an indirect ELISA assay based on the recombinant component Mus m 1.0102. The assay performed with adequate precision and reasonable diagnostic accuracy (AUC = 0.87) compared to a routine clinical diagnostic test that exploits the native allergen. Recombinant Mus m 1.0102 turned out to be a valuable tool to study the fine epitope mapping of specific IgE reactivity to the major allergen responsible for mouse allergy. We believe that advancing in its functional characterization will lead to the standardization of murine lipocalins and to the development of allergen-specific immunotherapy.

Differential critical residues on the overlapped region of the non-structural protein-1 recognized by flavivirus and dengue virus cross-reactive monoclonal antibodies

The non-structural protein-1 (NS1) of dengue virus (DENV) contributes to several functions related to dengue disease pathogenesis as well as diagnostic applications. Antibodies against DENV NS1 can cross-react with other co-circulating flaviviruses, which may lead to incorrect diagnosis. Herein, five anti-DENV NS1 monoclonal antibodies (mAbs) were investigated. Four of them (1F11, 2E3, 1B2, and 4D2) cross-react with NS1 of all four DENV serotypes (pan-DENV mAbs), whereas the other (2E11) also reacts with NS1 of other flaviviruses (flavi-cross-reactive mAb). The binding epitopes recognized by these mAbs were found to overlap a region located on the disordered loop of the NS1 wing domain (amino acid residues 104 to 123). Fine epitope mapping employing phage display technology and alanine-substituted DENV2 NS1 mutants indicates the critical binding residues W115, K116, and K120 for the 2E11 mAb, which are conserved among flaviviruses. In contrast, the critical binding residues of four pan-DENV mAbs include both flavi-conserved residues (W115 to G119) and DENV-conserved flanking residues (K112, Y113, S114 and A121, K122). Our results highlight DENV-conserved residues in cross-reactive epitopes that distinguish pan-DENV antibodies from the flavi-cross-reactive antibody. These antibodies can be potentially applied to differential diagnosis of DENV from other flavivirus infections.

High-Resolution Epitope Mapping and Affinity Binding Analysis Comparing a New Anti-Human LAG3 Rabbit Antibody Clone to the Commonly Used Mouse 17B4 Clone.

Lymphocyte activation gene 3 (LAG3) is a T cell inhibitory receptor that promotes tumor cell immune escape and is a potential target for cancer diagnostic and immunotherapeutic applications. We used automated capillary electrophoresis (ACE), surface plasmon resonance (SPR), and immunohistochemistry (IHC) to compare the binding characteristics of a new anti-LAG3 rabbit antibody clone, SP464, with the thirty-year old and extensively used anti-LAG3 mouse 17B4 clone. The rabbit SP464 clone exhibited between 20× to 30× greater binding to LAG3 than did the mouse 17B4 clone. Using these tools, we precisely mapped the relative locations of the epitopes of these two antibodies. The SP464 and 17B4 minimal epitopes were localized to separate, but overlapping, sub-fragments within the amino-terminal fifteen acids of the original thirty-mer peptide immunogen used to generate both antibodies. Application of this approach for quantifying the effects of alanine substitutions along the minimal SP464 epitope identified two amino acids essential for binding and four amino acids that likely contribute towards binding. Together, ACE, SPR, and IHC constitute a powerful orthologous approach for comparing antibody-binding characteristics and for fine mapping of linear epitopes within short immunogens. Our results indicate that the rabbit clone SP464 may be useful for assessing LAG3 expression.

Fine mapping of the antigenic epitopes of the Gc protein of Guertu virus.

Guertu virus (GTV), a newly discovered member of the genus Banyangvirus in the family Phenuiviridae, poses a potential health threat to humans and animals. The viral glycoprotein (GP) binds to host cell receptors to induce a neutralizing immune response in the host. Therefore, identification of the B-cell epitopes (BCEs) in the immunodominant region of the GTV Gc protein is important for the elucidation of the virus-host cell interactions and the development of GTV epitope assays and vaccines. In this study, an improved overlapping biosynthetic peptide method and rabbit anti-GTV Gc polyclonal antibodies were used for fine mapping of the minimal motifs of linear BCEs of the GTV Gc protein. Thirteen BCE motifs were identified from eleven positive 16mer-peptides, namely EGc1 (19KVCATTGRA27), EGc2 (58KKINLKCKK66), EGc3 (68SSYYVPDA75), EGc4 (75ARSRCTSVRR84), EGc5 (79CTSVRRCRWA88), EGc6 (90DCQSGCPS97), EGc7 (96PSHFTSNS103), EGc8 (115AGLGFSG121), EGc9 (148ENPHGVI154), EGc10 (179KVFHPMS185), EGc11 (230QAGMGVVG237), EGc12 (303RSHDSQGKIS312), and EGc13 (430DIPRFV435). Of these, 7 could be recognized by GTV IgG-positive sheep sera. Three-dimensional structural analysis revealed that all 13 BCEs were present on the surface of the Gc protein. Sequence alignment of the 13 BCEs against homologous proteins from 10 closely related strains of severe fever with thrombocytopenia syndrome virus from different geographical regions revealed that the amino acid sequences of EGc4, EGc5, EGc8, EGc11, and EGc12 were highly conserved, with 100% similarity. The remaining 8 epitopes (EGc1, EGc2, EGc3, EGc6, EGc7, EGc9, EGc10, and EGc13) showed high sequence similarity in the range of 71.43%-87.50%. These 13 BCEs of the GTV Gc protein provide a molecular foundation for future studies of the immunological properties of GTV glycoproteins and the development of GTV multi-epitope assays and vaccines.

Functional and in silico Characterization of Neutralizing Interactions Between Antibodies and the Foot-and-Mouth Disease Virus Immunodominant Antigenic Site.

Molecular knowledge of virus–antibody interactions is essential for the development of better vaccines and for a timely assessment of the spread and severity of epidemics. For foot-and-mouth disease virus (FMDV) research, in particular, computational methods for antigen–antibody (Ag–Ab) interaction, and cross-antigenicity characterization and prediction are critical to design engineered vaccines with robust, long-lasting, and wider response against different strains. We integrated existing structural modeling and prediction algorithms to study the surface properties of FMDV Ags and Abs and their interaction. First, we explored four modeling and two Ag–Ab docking methods and implemented a computational pipeline based on a reference Ag–Ab structure for FMDV of serotype C, to be used as a source protocol for the study of unknown interaction pairs of Ag–Ab. Next, we obtained the variable region sequence of two monoclonal IgM and IgG antibodies that recognize and neutralize antigenic site A (AgSA) epitopes from South America serotype A FMDV and developed two peptide ELISAs for their fine epitope mapping. Then, we applied the previous Ag–Ab molecular structure modeling and docking protocol further scored by functional peptide ELISA data. This work highlights a possible different behavior in the immune response of IgG and IgM Ab isotypes. The present method yielded reliable Ab models with differential paratopes and Ag interaction topologies in concordance with their isotype classes. Moreover, it demonstrates the applicability of computational prediction techniques to the interaction phenomena between the FMDV immunodominant AgSA and Abs, and points out their potential utility as a metric for virus-related, massive Ab repertoire analysis or as a starting point for recombinant vaccine design.

Phage-DMS: A Comprehensive Method for Fine Mapping of Antibody Epitopes.

SummaryUnderstanding the antibody response is critical to developing vaccine and antibody-based therapies and has inspired the recent development of new methods to isolate antibodies. Methods to define the antibody-antigen interactions that determine specificity or allow escape have not kept pace. We developed Phage-DMS, a method that combines two powerful approaches—immunoprecipitation of phage peptide libraries and deep mutational scanning (DMS)—to enable high-throughput fine mapping of antibody epitopes. As an example, we designed sequences encoding all possible amino acid variants of HIV Envelope to create phage libraries. Using Phage-DMS, we identified sites of escape predicted using other approaches for four well-characterized HIV monoclonal antibodies with known linear epitopes. In some cases, the results of Phage-DMS refined the epitope beyond what was determined in previous studies. This method has the potential to rapidly and comprehensively screen many antibodies in a single experiment to define sites essential for binding interactions.

Mapping of Recognition Sites of Monoclonal Antibodies Responsible for the Inhibition of Pneumolysin Functional Activity

The pathogenicity of many bacteria, including Streptococcus pneumoniae, depends on pore-forming toxins (PFTs) that cause host cell lysis by forming large pores in cholesterol-containing cell membranes. Therefore, PFTs-neutralising antibodies may provide useful tools for reducing S. pneumoniae pathogenic effects. This study aimed at the development and characterisation of monoclonal antibodies (MAbs) with neutralising activity to S. pneumoniae PFT pneumolysin (PLY). Five out of 10 produced MAbs were able to neutralise the cytolytic activity of PLY on a lung epithelial cell line. Epitope mapping with a series of recombinant overlapping PLY fragments revealed that neutralising MAbs are directed against PLY loops L1 and L3 within domain 4. The epitopes of MAbs 3A9, 6E5 and 12F11 located at L1 loop (aa 454–471) were crucial for PLY binding to the immobilised cholesterol. In contrast, the MAb 12D10 recognising L3 (aa 403–423) and the MAb 3F3 against the conformational epitope did not interfere with PLY-cholesterol interaction. Due to conformation-dependent binding, the approach to use overlapping peptides for fine epitope mapping of the neutralising MAbs was unsuccessful. Therefore, the epitopes recognised by the MAbs were analysed using computational methods. This study provides new data on PLY sites involved in functional activity.

Fine Epitope Mapping of the CD19 Extracellular Domain Promotes Design.

The B-cell surface protein CD19 is present throughout the cell life cycle and is uniformly expressed in leukemias, making it a target for chimeric antigen receptor engineered immune cell therapy. Identifying the sequence dependence of the binding of CD19 to antibodies empowers fundamental study and more tailored development of CD19-targeted therapeutics. To identify the antibody-binding epitopes on CD19, we screened a comprehensive single-site saturation mutation library of the human CD19 extracellular domain to identify mutations detrimental to binding FMC63-the dominant CD19 antibody used in chimeric antigen receptor development-as well as 4G7-2E3 and 3B10, which have been used in various types of CD19 research and development. All three antibodies had partially overlapping, yet distinct, epitopes near the published epitope of antibody B43. The FMC63 conformational epitope spans spatially adjacent, but genetically distant, loops in exons 3 and 4. The 3B10 epitope is a linear peptide sequence that binds CD19 with 440 pM affinity. Along with their primary goal of epitope mapping, the mutational tolerance data also empowered additional CD19 variant design and analysis. A designed CD19 variant with all N-linked glycosylation sites removed successfully bound antibody in the yeast display context, which provides a lead for aglycosylated applications. Screening for thermally stable variants identified mutations to guide further CD19 stabilization for fusion protein applications and revealed evolutionary affinity-stability trade-offs. These fundamental insights into CD19 sequence-function relationships enhance our understanding of antibody-mediated CD19-targeted therapeutics.

Fine epitope mapping of glycoprotein Gn in Guertu virus.

Guertu virus (GTV) is a tick-borne phleboviruses (TBPVs) which belongs to the genus Banyangvirus in the family of Phenuiviridae. In vitro and in vivo studies of GTV demonstrated that it was able to infect animal and human cell lines and could cause pathological lesions in mice. Glycoproteins (GP, including Gn and Gc) on the surface of Guertu virus (GTV) could bind to receptors on host cells and induce protective immunity in the host, but knowledge is now lacking on the information of B cell epitopes (BCEs) present on GTV-GP protein. The aim of this study was to identify all BCEs on Gn of the GTV DXM strain using rabbit pAbs against GTV-Gn. Seven fine BCEs and two antigenic peptides (APs) from nine reactive 16mer-peptides were identified, which are EGn1 (2PIICEGLTHS11), EGn2 (135CSQDSGT141), EGn3 (165IP EDVF170), EGn4 (169VFQEL K174), EGn5 (187IDGILFN193), EGn6 (223QTKWIQ228), EGn7 (237CHKDGIGPC245), AP-8 (299GVRVRPKCYGFSRMMA314) and AP-9 (355CASH FCSSAESGKKNT370), of which six of mapped BCEs were recognized by the IgG-positive sheep serum obtained from sheep GTV-infected naturally. Multiple sequence alignments (MSA) based on each mapped BCE motif identified that the most of identified BCEs and APs are highly conserved among 10 SFTSV strains from different countries and lineages that share relatively close evolutionary relationships with GTV. The fine epitope mapping of the GTV-Gn would provide basic data with which to explore the GTV-Gn antigen structure and pathogenic mechanisms, and it could lay the foundation for the design and development of a GTV multi-epitope peptide vaccine and detection antigen.

Epitope mapping and cellular localization of swine acute diarrhea syndrome coronavirus nucleocapsid protein using a novel monoclonal antibody

A swine acute diarrhea syndrome coronavirus (SADS-CoV) that causes severe diarrhea in suckling piglets was identified in Southern China in 2017. To develop an antigen that is specific, sensitive, and easy to prepare for serological diagnosis, antigenic sites in the SADS-CoV nucleocapsid (N) protein were screened. We generated and characterized an N-reactive monoclonal antibody (mAb) 3E9 from mice immunized with recombinant N protein. Through fine epitope mapping of mAb 3E9 using a panel of eukaryotic-expressed polypeptides with GFP-tags, we identified the motif 343DAPVFTPAP351 as the minimal unit of the linear B-cell epitope recognized by mAb 3E9. Protein sequence alignment indicated that 343DAPVFTPAP351 was highly conserved in different SADS-CoV strains and SADS-related coronaviruses from bat, with one substitution in this motif in HKU2-related bat coronavirus. Using mAb 3E9, we observed that N protein was expressed in the cytoplasm and was in the nucleolus during SADS-CoV replication. N protein was immunoprecipitated from SADS-CoV-infected Vero E6 cells. Taken together, our results indicated that 3E9 mAb could be a useful tool to investigate the structure and function of N protein during viral replication.

ExMS2: An Integrated Solution for Hydrogen-Deuterium Exchange Mass Spectrometry Data Analysis.

Hydrogen-deuterium exchange mass spectrometry (HDX MS) has become an important technique for the analysis of protein structure and dynamics. Data analysis remains a bottleneck in the workflow. Sophisticated computer analysis is required to scan through the voluminous MS output in order to find, identify, and validate many partially deuterated peptides, elicit the HDX information, and extend the results to higher structural resolution. We previously made available two software suites, ExMS for identification and analysis of peptide isotopic envelopes in the HDX MS raw data and HDsite for residue-level resolution. Further experience has led to advances in the usability and performance of both programs. Also, newly added modules deal with ETD/ECD analysis, multimodal mass spectra analysis, and presentation options. These advances have been integrated into a stand-alone software solution named ExMS2. The package has been successfully tested by many workers in fine scale epitope mapping, in protein folding studies, and in dissecting structure and structure change of large protein complexes. A description and tutorial for this major upgrade are given here.

Microbial exposure and autoimmunity in the etiopathogenesis of Sarcoidosis

Abstract Sarcoidosis is an enigmatic inflammatory disease affecting multiple organs such as the lungs, lymph nodes, brain, heart, and skin. It is characterized by the occurrence of multiple non-caseating granulomas comprised of macrophages, giant cells, and CD4 T cells. Although genetic and environmental factors increase the risk for sarcoidosis, the etiology remains unknown. Recent studies have shown that HLA-DR3+ sarcoidosis patients harbor CD4+ T cells reactive with a vimentin peptide 424–443. However, whether autoimmunity is a cause or consequence of the disease is not known. This study was undertaken to investigate the mechanisms responsible for activation of vimentin reactive T cells and determine their pathogenic role in sarcoidosis. For this purpose, we immunized the NOD-DR3 mice with the vimentin peptide 424–443 and induced a robust T cell response against the peptide. Fine epitope mapping using T cell hybridomas reactive with the peptide showed that both N-terminal and C-terminal amino acids were critical for T cell activation. Homology searches of microbial protein database identified a 100% homologous sequence in a protein from Klebsiella pneumoniae. Immunization of mice with this peptide, followed by intravenous transfer of peptide-coupled agarose beads induced granulomatous lesions in the lungs. Immunohistochemical analysis of these lesions showed the presence of CD4+ T cells, macrophages, and an upregulated MHC-II expression. In summary, our data demonstrate for the first time that autoimmunity is involved in the pathogenesis of sarcoidosis. Furthermore, the demonstration of molecular mimicry between a lung infecting microbe and vimentin might explain the role of microbial exposures as a risk factor for sarcoidosis.

Fine epitope mapping of a human disulphide-stabilized diabody against fibroblast growth factor-2.

The human fibroblast growth factor-2 (FGF-2) highly expressed in tumours is an important factor to promote tumour angiogenesis and lymphangiogenesis. A disulphide-stabilized diabody (ds-Diabody) could specifically target FGF-2 and show its advantages in inhibition of tumour angiogenesis and growth. It is very important for antibody drugs to confirm the fine epitope. Here, theoretical structure models of FGF-2 and antibody were built by homology modelling. The amino acid residues in the interaction interface of antigen and antibody were analysed by molecular docking. The potential epitope was predicted by homology modelling and molecular docking of antigen-antibody and site-directed mutation assays of alanine scanning. The predicted epitope was verified by antigen mutagenesis and enzyme-linked immunosorbent assay (ELISA). The epitope mapping assay showed that the epitope of ds-Diabody against FGF-2 was defined by the discontinuous sites including six amino acid residues (P23, Q65, R69, G70, Y82 and R118). The results showed that the epitope was localized in the interaction interface of FGF-2 and ds-Diabody. The fine epitope mapping provided the important information for understanding the inhibition activity of ds-Diabody against FGF-2 and helping in the further development of ds-Diabody against FGF-2 as a potentially promising antibody drug for future cancer therapy.

Production, characterization, and epitope mapping of a monoclonal antibody against genotype VII Newcastle disease virus V protein

Newcastle disease virus (NDV) V protein is crucial for viral interferon (IFN) antagonism and virulence, determining its host range restriction. However, little information is available on the B cell epitopes of V protein and the subcellular movement of V protein in the process of NDV infection. In this study, the monoclonal antibody (mAb) clone 3D7 against genotype VII NDV V protein was generated by immunizing mice with a purified recombinant His-tagged carboxyl-terminal domain (CTD) region of V protein. Fine epitope mapping analysis and B-cell epitope prediction indicated that mAb 3D7 recognized a linear epitope 152RGPAELWK159, which is located in the V protein CTD region. Sequence alignment showed that the mAb clone 3D7-recognized epitope is highly conserved among Class II genotype VII NDV strains, but not among other genotypes, suggesting it could serve as a genetic marker to differentiate NDV genotypes. Furthermore, the movement of V protein during NDV replication in infected cells were determined by using this mAb. It was found that V protein localized around the nucleus during virus replication. The establishment of V protein-specific mAb and identification of its epitope extend our understanding of the antigenic characteristics of V protein and provide a basis for the development of epitope-based diagnostic assays.

Common antibody dependent cell mediated cytotoxicity (ADCC) antibody epitopes of HIV-1 CRF01_AE Env and Gag in early HIV-1 infected individuals

There have been a few studies aimed at identifying epitopes of ADCC-inducing antibodies when compared to those of neutralizing antibodies and cytotoxic T lymphocytes against a variety of HIV-1 clades. To map the common ADCC epitopes of HIV-1 CRF01_AE. We screened 65 sera of confirmed early HIV-1 CRF01_AE infected individuals for ADCC antibody against gp120 utilizing an EGFP-CEM-NKr flow cytometric assay. Sera with high ADCC antibody were then examined against ADCC epitopes using the complete HIV-1 CRF01_AE gp160- and subtype A Gag-overlapping peptide sets which were divided into 7 pools:E1-E7 and 5 pools:G1-G5, respectively. Each positive peptide pool was further investigated for fine ADCC epitope mapping using matrix formats. Twenty, 25 and 20 sera demonstrated the high-, medium- and low-ADCC antibody activities against gp120, respectively. Interestingly, 11 Env- and 6 Gag-peptides of pools E3, E4, E7 and pools G1, G2, G4 with high ADCC responses were also responded by at least 20%, 12% and 5%, 10% of medium- and low-ADCC antibody sera, respectively. These eleven common Env ADCC epitopes were localized at C2-V3-C3-V4 regions of gp120 and cytoplasmic tail of gp41 while six common Gag ADCC epitopes were localized at p17-p24-p2 regions. Although the degree of ADCC antibody responses to the gp120 protein varied from high to low, there were certain consensus Env and Gag peptides that could induce the ADCC antibody responses of 21.54-58.46% and 23.08-41.54%, respectively of the early infected individuals. This epitope information should be useful as the new antibody-based vaccine immunogens.

Epitope mapping and characterization of a novel Nsp10-specific monoclonal antibody that differentiates genotype 2 PRRSV from genotype 1 PRRSV

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of PRRS, has two distinct and highly diverse genotypes (genotype 1 and genotype 2) in the field. Accurate diagnosis and differentiation of the two genotypes of PRRSV are critical to the effective prevention and control of PRRS. The non-structural protein 10 (Nsp10) plays a vital role in viral replication and is one of the most conserved proteins of PRRSV, thus constituting a good candidate for PRRSV diagnosis.ResultsIn this study, we generated a monoclonal antibody (mAb) 4D9 against Nsp10 by immunizing BALB/c mice with purified recombinant Nsp10 expressed by an Escherichia coli system. Through fine epitope mapping of mAb 4D9 using a panel of eukaryotic expressed polypeptides with GFP-tags, we identified the motif 286AIQPDYRDKL295 as the minimal unit of the linear B-cell epitope recognized by mAb 4D9. Protein sequence alignment indicated that 286AIQPDYRDKL295 was highly conserved in genotype 2 PRRSV strains, whereas genotype 1 PRRSV strains had variable amino acids in this motif. Furthermore, a mutant of the motif carrying two constant amino acids of genotype 1 PRRSV, Cys290 and Glu293, failed to react with mAb 4D9. More importantly, the mAb 4D9 could differentiate genotype 2 PRRSV strains from genotype 1 PRRSV strains using Western blotting and immunofluorescence analysis.ConclusionOur findings suggest that Nsp10-specific mAb generated in this study could be a useful tool for basic research and may facilitate the establishment of diagnostic methods to discriminate between genotype 1 and genotype 2 PRRSV infection.

Fine Epitope Mapping of Monoclonal Antibodies to the DNA Repair Protein, RadA.

Repair of DNA damage is vital to the health and survival of all organisms. In Escherichia coli, a protein known as RadA (or Sms) participates in recombinational repair, a process that uses an undamaged DNA strand in one DNA duplex to fill a gap in a homologous DNA strand in a sister DNA duplex. In a prior report, we described the production of monoclonal antibodies (MAbs) specific for RadA. Here, we investigated the epitopes recognized by two of the antibodies, MAbs 6F5 and 2A2. Premature stop codons (ochre mutations) were introduced into the radA gene at selected sites, and the truncated RadA proteins were probed by western blotting. Deletion of as few as four amino acids (457-460) from the C-terminus of RadA significantly increased the sensitivity of E. coli to ultraviolet (UV) radiation and abolished recognition of RadA by MAb 6F5. Single alanine substitutions made between positions 443-460 also adversely affected the ability of MAb 6F5 to bind to RadA, further supporting the idea that MAb 6F5 is specific for the RadA C-terminus. An ochre mutation at position 258 abolished the recognition of RadA by MAb 2A2, whereas an ochre mutation at position 279 did not, suggesting that MAb 2A2 binds to an epitope between residues 258 and 279. MAb 2A2 recognition of RadA was destroyed by endoproteinase glu-C cleavage of RadA at position 266, and by a single alanine substitution at position 265. In a competitive enzyme-linked immunosorbent assay (ELISA), a synthetic peptide comprising residues 263-273 of RadA blocked MAb 2A2 recognition of immobilized full-length RadA by more than 97%. We infer from our results that MAb 6F5 binds to the extreme C-terminus of RadA and that MAb 2A2 is specific for an epitope within positions 263-273.

Humanized monoclonal antibody armanezumab specific to N-terminus of pathological tau: characterization and therapeutic potency

BackgroundThe experience from clinical trials indicates that anti-Aβ immunotherapy could be effective in early/pre-clinical stages of AD, whereas at the late stages promoting the clearing of Aβ alone may be insufficient to halt the disease progression. At the same time, pathological tau correlates much better with the degree of dementia than Aβ deposition. Therefore, targeting pathological tau may provide a more promising approach for the treatment of advanced stages of AD. Recent data demonstrates that the N-terminal region of tau spanning aa 2–18 termed “phosphatase activation domain” that is normally hidden in the native protein in ‘paperclip’-like conformation, becomes exposed in pathological tau and plays an essential role in the inhibition of fast axonal transport and in aggregation of tau. Hence, we hypothesized that anti-Tau2–18 monoclonal antibodies (mAb) may recognize pathological, but not normal tau at very early stages of tauopathy and prevent or decrease the aggregation of this molecule.MethodsMouse mAbs were generated using standard hybridoma methodology. CDR grafting was used for humanization of mouse mAb. Humanized mAb (Armanezumab) was characterized and tested in vitro/ex vivo/in vivo using biochemical and immunological methods (HPLC, Biacore, ELISA, IHC, FRET, etc.). Stable DG44 cell line expressing Armanezumab was generated by clone selection with increased concentrations of methotrexate (MTX).ResultsA panel of mouse mAbs was generated, clone 1C9 was selected based on binding to pathological human tau with high affinity and humanized. Fine epitope mapping revealed conservation of the epitope of human tau recognized by the parent murine mAb and Armanezumab. Importantly, Armanezumab (i) bound to tau with high affinity as determined by Biacore; (ii) bound pathological tau in brains from AD, FTD and Pick’s disease cases; (iii) inhibited seeding effect of aggregated tau from brain lysate of P301S Tg mice; (iv) inhibited cytotoxic effect of tau oligomers; (v) reduced total tau (HT7) and AT100, PHF1, AT8, AT180, p212, p214-positive tau species in brains of tau transgenic mice after intracranial injection. A stable CHO cell line producing >1.5 g/l humanized mAb, Armanezumab was generated.ConclusionThese findings suggest that Armanezumab could be therapeutic in clinical studies for treatment of AD.